Hepatitis c virus immunogenic compositions and methods of use thereof

ABSTRACT

The present disclosure provides an immunogenic composition comprising: a) a hepatitis C virus (HCV) heterodimeric polypeptide that includes HCV E1 and E2 polypeptides; b) a T-cell epitope polypeptide comprising a T-cell epitope present in an HCV protein other than E1 and E2; and c) a pharmaceutically acceptable excipient. The present disclosure provides a method of inducing an immune response, in an individual, to an HCV polypeptide. The present disclosure provides an immunogenic composition comprising: a) a polypeptide that comprises one or more T-cell epitopes present in an HCV protein other than E1 and E2; and b) a pharmaceutically acceptable excipient.

CROSS-REFERENCE

This application is a national stage filing under 35 U.S.C. § 371 ofPCT/IB2017/055714, filed Sep. 21, 2017, which claims the benefit of U.S.Provisional Patent Application No. 62/397,763, filed Sep. 21, 2016,which applications are incorporated herein by reference in theirentirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “UALB-034WO_SEQLISTING_ST25.txt” created on Sep. 18, 2017 and having a size of 762 KB.The contents of the text file are incorporated by reference herein intheir entirety.

INTRODUCTION

Hepatitis C virus (HCV) is a blood-borne pathogen that is estimated toinfect 150-200 million people worldwide. Infection by HCV may benon-symptomatic, and can be cleared by patients, sometimes withoutmedical intervention. However, the majority of patients develop achronic HCV infection, which may lead to liver inflammation, scarring,and even to liver failure or liver cancer. In the United States alone,over 3 million people have a chronic infection.

The HCV virion contains a positive-sense single stranded RNA genome ofabout 9.5 kb. The genome encodes a single polyprotein of 3,010 to 3,030amino acids. The structural proteins comprise a core protein forming theviral nucleocapsid and two envelope glycoproteins, E1 and E2.

A vaccine based on the recombinant envelope glycoproteins from a singlegenotype 1a strain (HCV-1) protected chimpanzees from chronic infectionfollowing homologous and heterologous genotype 1a (gt1a) viral challenge(reviewed in Houghton, M Immunol Rev 2011). Antisera from the immunizedchimpanzees were shown to exhibit in vitro cross-neutralizing activity(Meunier et al. (2011) J. Infect. Dis. 204:1186). A phase I clinicaltrial was conducted in human volunteers with a similar antigen (Frey etal. (2010) Vaccine 28:6367). Antisera from selected vaccinatedindividuals were similarly capable of neutralizing chimeric cellculture-derived viruses (HCVcc) expressing the structural proteins ofstrains representing all 7 major HCV genotypes in vitro (Law et al.(2013) PLoS One 8:e59776) and to be able to compete with the binding ofnumerous discrete monoclonal antibodies with broad cross-neutralizingactivities (Wong et al. (2014) J. Virol. 88:14278).

There is a need in the art for compositions and methods for inducingimmune responses to HCV.

SUMMARY

The present disclosure provides an immunogenic composition comprising:a) i) a hepatitis C virus (HCV) heterodimeric polypeptide that includesHCV E1 and E2 polypeptides; ii) an HCV E1 polypeptide; or iii) an HCV E2polypeptide; b) a heterologous polypeptide (also referred to herein as a“T-cell epitope polypeptide” or an “HCV T-cell epitope polypeptide”)comprising T-cell epitopes (e.g., CD4⁺ and CD8⁺ T-cell epitopes that areconserved among heterogeneous HCV genotypes and that are presentedthrough multiple HLA alleles common within the human population) presentin an HCV protein other than E1 and E2; and c) a pharmaceuticallyacceptable excipient. The present disclosure provides a method ofinducing an immune response, in an individual, to an HCV polypeptide.The present disclosure provides an immunogenic composition comprising:a) a polypeptide that comprises one or more T-cell epitopes (e.g., CD4⁺and CD8⁺ T-cell epitopes that are conserved among heterogeneous HCVgenotypes and that are presented through multiple HLA alleles commonwithin the human population) present in an HCV protein other than E1 andE2; and b) a pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C provide an amino acid sequence alignment of examples of thecore-E1-E2 coding regions of a HCV genotype 1 virus, specificallyrepresentative HCV 1A, 1B and 1C genotypes. Genbank database sequencesfor the coding region core-E1-E2 were aligned using Geneious softwarev5.6.4. Numbering of amino acids is according to strain NP_671941 (H77).Consensus: SEQ ID NO:1; AVI1a129: SEQ ID NO:2; NP_671491 (H77): SEQ IDNO:3; EU155269: SEQ ID NO:4; EU781810: SEQ ID NO:5; EU781771: SEQ IDNO:6; AB250610: SEQ ID NO:7; EU781752: SEQ ID NO:8; EU781759: SEQ IDNO:9; EF407439: SEQ ID NO:10; EF407427: SEQ ID NO:11; EU362905: SEQ IDNO:12; EF407413: SEQ ID NO:13; EU781808: SEQ ID NO:14; EU78170: SEQ IDNO:15; AJ238799 (Con1): SEQ ID NO:16; AAK97744: SEQ ID NO:17; AF139594:SEQ ID NO:18; AF176573: SEQ ID NO:19; BAA19625: SEQ ID NO:20; BAA25076:SEQ ID NO:21; BAC54896: SEQ ID NO:22; BAD91386: SEQ ID NO:23; BAF46764:SEQ ID NO:24; BAG30950: SEQ ID NO:25; CAB41951: SEQ ID NO:26; AAK95832:SEQ ID NO:27; AAT69968: SEQ ID NO:28; and BAA03581: SEQ ID NO:29.

FIG. 2A-2C provide an alignment of amino acid sequences of thecore-E1-E2 coding region of representative HCV 2A and HCV2B subtypes.Genbank database sequences for the coding region core-E1-E2 were alignedusing Geneious software v5.6.4. The amino acid numbering depicted is inaccordance to the common HCV strains: ABO47639 (JFH1) and HPCJ8G-J8 (J8)for HCV2A and HCV2B, respectively. AB047639 (JFH1): SEQ ID NO:30;AB047645: SEQ ID NO:31; AF169003: SEQ ID NO:32; AF169005: SEQ ID NO:33;AF238482: SEQ ID NO:34; AY746460: SEQ ID NO:35; HPCPOLP: SEQ ID NO:36;NC_009823: SEQ ID NO:37; HPCJ8G HC-J8: SEQ ID NO:38; AB030907: SEQ IDNO:39; AY232730: SEQ ID NO:40; AY232747: SEQ ID NO:41; and DQ430817: SEQID NO:42.

FIG. 3A-3C provide an amino acid sequence alignment of the core-E1-E2coding region for representative HCV 3A, 3B and 3K genotypes. Genbankdatabase sequences for the coding region core-E1-E2 were aligned usingGeneious software v5.6.4. Consensus: SEQ ID NO:43; AVI3a177: SEQ IDNO:44; YP_0014696: SEQ ID NO:45; CAA54244: SEQ ID NO:46; AAC03058: SEQID NO:47; AAY29642: SEQ ID NO:48; ABD85062: SEQ ID NO:49; ABD85063: SEQID NO:50; ABD97104: SEQ ID NO:51; BAA06044: SEQ ID NO:52; BAA08372: SEQID NO:53; and BAA09890: SEQ ID NO:54.

FIG. 4A-4B provide an amino acid sequence of the core-E1-E2 codingregion for HCV genotype 7a. Amino acid sequence for the coding regioncore-E1-E2 of genotype 7a (isolate QC69; Genbank: ABN05226.1; SEQ IDNO:55) is shown according to the numbering scheme of the referencestrain, NP_671941 (H77).

FIG. 5A-5C provide amino acid sequences of immunoglobulin Fc regions forGenBank 3S7G_A Homo sapiens IgG1 Fc: SEQ ID NOs.:140; GenBank AAN76044Homo sapiens IgG2 Fc: SEQ ID NO: 141; GenBank AAW65947 Homo sapiens IgG3Fc: SEQ ID NO:142; GenBank AAA52770 Homo sapiens IgD Fc: SEQ ID NO:143;GenBank 0308221A Homo sapiens IgM Fc: SEQ ID NO:144; GenBank P01876 Homosapiens Iga Fc: SEQ ID NO:145; GenBank IF6A_B Homo sapiens IgE Fc: SEQID NO:146; and GenBank P01861 Homo sapiens IgG4 Fc: SEQ ID NO:147.

FIG. 6 presents Table 1, which provides conserved regions based onconserved CD4 epitopes (CD4⁺ T cell epitopes) conserved among HCVgenotypes. Top to Bottom: SEQ ID NOs.:148-158.

FIG. 7 presents Table 2, which provides the number of located HCV CD8⁺ Tcell epitopes and anchor positions for common human leukocyte antigen(HLA)-I Alleles in the United States.

FIG. 8 presents Table 3, which provides conserved regions based on CD8epitopes (CD8⁺ T cell epitopes) conserved among HCV genotypes. Top toBottom: SEQ ID NOs.:159-168.

FIG. 9A-9B provide a list of CD4 and CD8 epitopes that are conservedamong HCV genotypes 1a, 1b, 2a, 2b, and 3.

FIG. 10A-10D provide amino acid sequences of examples of polypeptidescomprising multiple T-cell epitopes (TP29: SEQ ID NO:94; TP50: SEQ IDNO:99; TP52: SEQ ID NO:95; TP70: SEQ ID NO:96; TP100: SEQ ID NO:97;TP171: SEQ ID NO:89; TP228: SEQ ID NO:81; TP553: SEQ ID NO:100; TP778:SEQ ID NO:101; and TP1985: SEQ ID NO:102). The start and end amino acidsare based on the sequence designated “Consensus” in FIG. 12A-12L. TheT-cell epitopes contained within each polypeptide are provided; theT-cell epitope designations correspond to those presented in FIG.11A-11N.

FIG. 11A-11N provide consensus amino acid sequences of HCV polypeptides;and depict the locations of T-cell epitopes (SEQ ID NO:169).

FIG. 12A-12L provide consensus amino acid sequences of HCV polypeptides(SEQ ID NOs.:170-181).

DEFINITIONS

The term “hepatitis C virus” (“HCV”), as used herein, refers to any oneof a number of different genotypes and isolates of hepatitis C virus.Thus, “HCV” encompasses any of a number of genotypes, subtypes, orquasispecies, of HCV, including, e.g., genotype 1, 2, 3, 4, 6, 7, etc.and subtypes (e.g., 1a, 1b, 2a, 2b, 3a, 4a, 4c, etc.), and quasispecies.Representative HCV genotypes and isolates include: the “Chiron” isolateHCV-1, H77, J6, Con1, isolate 1, BK, EC1, EC10, HC-J2, HC-J5; HC-J6,HC-J7, HC-J8, HC-JT, HCT18, HCT27, HCV-476, HCV-KF, “Hunan”, “Japanese”,“Taiwan”, TH, type 1, type 1a, H77 type 1b, type 1c, type 1d, type 1e,type 1f, type 10, type 2, type 2a, type 2b, type 2c, type 2d, type 2f,type 3, type 3a, type 3b, type 3g, type 4, type 4a, type 4c, type 4d,type 4f, type 4h, type 4k, type 5, type 5a, type 6 and type 6a.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, non-human primates (e.g., simians), equines (e.g., horses),rodents (e.g., rats; mice), and humans.

As used herein, the term “isolated,” in reference to a polypeptide,refers to a polypeptide that is in an environment different from that inwhich the polypeptide naturally occurs. An isolated polypeptide can bepurified. By “purified” is meant a compound of interest (e.g., apolypeptide) has been separated from components that accompany it innature. “Purified” can also be used to refer to a polypeptide separatedfrom components that can accompany it during production of thepolypeptide (e.g., during synthesis in vitro, etc.). In someembodiments, a polypeptide (or a mixture of polypeptides) issubstantially pure when the polypeptide (or mixture of polypeptides) isat least 60% or at least 75% by weight free from organic molecules withwhich it is naturally associated or with which it is associated duringproduction. In some embodiments, the polypeptide is from 30% to 60%pure. In some embodiments, the polypeptide (or mixture of polypeptides)is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%,at least 95%, or at least 99%, by weight, pure. For example, in someembodiments, an E1 or an E2 polypeptide (or a mixture of E1 and E2polypeptides, e.g., an E1/E2 heterodimer) is substantially pure when theE1 or E2 polypeptide (or mixture of E1 and E2 polypeptides) is at least60% or at least 75% by weight free from organic molecules with which thepolypeptide(s) is naturally associated or with which it is associatedduring production. In some embodiments, the E1 or E2 polypeptide (ormixture of E1 and E2 polypeptides) is at least 60%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%, byweight, pure. In some embodiments, where a composition comprises an E2polypeptide, the E2 polypeptide is at least 60%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99%, byweight, pure. In some embodiments, where a composition comprises anE1/E2 heterodimeric complex polypeptide, the E1/E2 heterodimeric complexpolypeptide is at least 60%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or at least 99%, by weight, pure. In someembodiments, where a composition comprises a T-cell epitope polypeptide,the T-cell epitope polypeptide is at least 60%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99%, byweight, pure.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases. In some cases, apolynucleotide is RNA. In some cases, a polynucleotide is DNA. A“polynucleotide” includes a nucleic acid that is incorporated into aviral vector or a bacterial vector.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term “polypeptide”includes glycosylated polypeptides.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, where“heterologous” is used in the context of a polypeptide, where thepolypeptide includes operably linked amino acid sequences that can bederived from one or more different polypeptides, e.g., amino acidsequences that are not operably linked to the polypeptide in nature. Asanother example, where a composition comprises an HCV E1/E2 heterodimerand a “heterologous” polypeptide, the “heterologous polypeptide is apolypeptide other than HCV E1 or HCV E2. As another example, where acomposition comprises an HCV E1 polypeptide and a “heterologous”polypeptide, the “heterologous polypeptide is a polypeptide other thanHCV E1. As another example, where a composition comprises an HCV E2polypeptide and a “heterologous” polypeptide, the “heterologouspolypeptide is a polypeptide other than HCV E2.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aT-cell epitope” includes a plurality of such epitopes and reference to“the E1/E2 heterodimer” includes reference to one or more E1/E2heterodimers and equivalents thereof known to those skilled in the art,and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides an immunogenic composition comprising;a) an HCV heterodimeric polypeptide that includes HCV E1 and E2polypeptides; b) a heterologous polypeptide (a T-cell epitopepolypeptide) comprising T-cell epitopes present in an HCV protein otherthan E1 and E2; and c) a pharmaceutically acceptable excipient. Thepresent disclosure provides an immunogenic composition comprising; a) anHCV E2 polypeptide; b) a heterologous polypeptide (a T-cell epitopepolypeptide) comprising T-cell epitopes present in an HCV protein otherthan E1 and E2; and c) a pharmaceutically acceptable excipient. Thepresent disclosure provides an immunogenic composition comprising; a) anHCV E1 polypeptide; b) a heterologous polypeptide (a T-cell epitopepolypeptide) comprising T-cell epitopes present in an HCV protein otherthan E1 and E2; and c) a pharmaceutically acceptable excipient. T-cellepitopes that are present in a heterologous polypeptide suitable forinclusion in an immunogenic composition of the present disclosureinclude CD4⁺ and CD8⁺ T-cell epitopes that are conserved amongheterogeneous HCV genotypes and that are presented through multiple HLAalleles common within the human population. The present disclosureprovides a method of inducing an immune response, in an individual, toan HCV polypeptide. The present disclosure provides an immunogeniccomposition comprising: a) polypeptide comprising T-cell epitopespresent in an HCV protein other than E1 and E2; and b) apharmaceutically acceptable excipient. T-cell epitopes that are presentin the polypeptide include CD4⁺ and CD8⁺ T-cell epitopes that areconserved among heterogeneous HCV genotypes and that are presentedthrough multiple HLA alleles common within the human population.

In some cases, an immunogenic composition of the present disclosurecomprises, as separate entities: a) an HCV E1/E2 heterodimer; and b) aheterologous polypeptide comprising a T-cell epitope present in an HCVprotein other than E1 and E2. In some cases, an immunogenic compositionof the present disclosure comprises, as separate entities: a) an HCV E2polypeptide; and b) a heterologous polypeptide comprising a T-cellepitope present in an HCV protein other than E1 and E2. In some cases,an immunogenic composition of the present disclosure comprises, asseparate entities: a) an HCV E1 polypeptide; and b) a heterologouspolypeptide comprising a T-cell epitope present in an HCV protein otherthan E1 and E2.

As noted above, T-cell epitopes that are present in a heterologouspolypeptide suitable for inclusion in an immunogenic composition of thepresent disclosure include CD4⁺ and CD8⁺ T-cell epitopes that areconserved among heterogeneous HCV genotypes and that are presentedthrough multiple HLA alleles common within the human population. Thus, aheterologous polypeptide suitable for inclusion in an immunogeniccomposition of the present disclosure comprises multiple (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10) CD4⁺ and CD8⁺ T-cell epitopes thatare conserved among heterogeneous HCV genotypes and that are presentedthrough multiple HLA alleles common within the human population.

A suitable heterologous polypeptide comprises an amino acid sequencehaving at least 20% amino acid sequence identity to a polypeptidedepicted in any one of FIGS. 9A, 9B, 10A-10D, and 11A-11N. Theheterologous polypeptide can be expressed in any suitable host celle.g., a bacterial host cell, a yeast host cell, an insect host cell, amammalian host cell) as a separate polypeptide, then combined with aE1/E2 heterodimer, an E2 polypeptide, or an E1 polypeptide, to form andimmunogenic composition. The heterologous polypeptides serve to elicitbroad spectrum CD4⁺ and CD8⁺ T cell responses to multiple HCV genotypesbecause the heterologous polypeptides have been selected to contain aplurality of T cell epitopes that are highly conserved among thehepacivirus genus, many of which are immunodominant. The heterologouspolypeptides also contain T cell epitopes presented by various MHCalleles common in the human population. The E1/E2 antigens will alsoelicit cross-reactive T cell responses; however, the heterologouspolypeptides will elicit broader T cell responses that arecross-reactive with multiple HCV genotypes in the general humanpopulation. Both neutralizing antibodies and T cell responses are knownto be protective against HCV; thus, this combination of antigens,optionally along with a suitable adjuvant (e.g., AS01 or MF59 orAlum/MPL) will optimize the protective effects of a HCV vaccine.

The heterologous polypeptides may be expressed alone (e.g., without anyheterologous polypeptide appended thereto), and then purifiedconventionally. Alternatively, the heterologous polypeptides can beexpressed downstream of, or upstream of, an immunoglobulin (Ig) Fcfragment (or other affinity tag) separated by a protease cleavage site(e.g., a Precision protease cleavage site) and then purified. Theheterologous polypeptides can also be chemically-synthesised.

Compositions Comprising: A) an HCV E1/E2 Heterodimer, an HCV E2Polypeptide, or an HCV E1 Polypeptide; and B) a Heterologous PolypeptideComprising a T-Cell Epitope

The present disclosure provides an immunogenic composition comprising:a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising: i) anHCV E1 polypeptide; and ii) an HCV E2 polypeptide; b) a heterologouspolypeptide comprising a T-cell epitope present in an HCV protein otherthan E1 and E2; and c) a pharmaceutically acceptable carrier. In somecases, the immunogenic composition comprises an adjuvant. The presentdisclosure provides an immunogenic composition comprising: a) an HCV E2polypeptide; b) a heterologous polypeptide comprising a T-cell epitopepresent in an HCV protein other than E1 and E2; and c) apharmaceutically acceptable carrier. The present disclosure provides animmunogenic composition comprising: a) an HCV E1 polypeptide; b) aheterologous polypeptide comprising a T-cell epitope present in an HCVprotein other than E1 and E2; and c) a pharmaceutically acceptablecarrier. As noted above, T-cell epitopes that are present in aheterologous polypeptide suitable for inclusion in an immunogeniccomposition of the present disclosure include CD4⁺ and CD8⁺ T-cellepitopes that are conserved among heterogeneous HCV genotypes and thatare presented through multiple HLA alleles common within the humanpopulation. Thus, a heterologous polypeptide suitable for inclusion inan immunogenic composition of the present disclosure comprises multiple(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) CD4⁺ and CD8⁺ T-cellepitopes that are conserved among heterogeneous HCV genotypes and thatare presented through multiple HLA alleles common within the humanpopulation. In some cases, the immunogenic composition comprises anadjuvant.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces an immuneresponse in the individual to one or more HCV genotypes. In some cases,an immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response in theindividual to one or more HCV genotypes, where the immune response isgreater than the immune response induced by administration of a controlcomposition comprising the HCV E1/E2 heterodimer (or E1 polypeptide, orE2 polypeptide) but lacking the heterologous polypeptide.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces CD8⁺ CTLsspecific for HCV, where the number of HCV-specific CD8⁺ CTLs induced isat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the number of HCV-specific CD8⁺ CTLs induced byadministration of a control composition (e.g., a composition comprisingthe HCV E1/E2 heterodimer but lacking the heterologous polypeptide; acomposition comprising an E1 polypeptide but lacking the heterologouspolypeptide; a composition comprising an E2 polypeptide but lacking theheterologous polypeptide).

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces CD4⁺ T cellsspecific for HCV, where the number of HCV-specific CD4⁺ T cells inducedis at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the number of HCV-specific CD4⁺ T cells induced byadministration of a control composition (e.g., a composition comprisingthe HCV E1/E2 heterodimer but lacking the heterologous polypeptide; acomposition comprising an E1 polypeptide but lacking the heterologouspolypeptide; a composition comprising an E2 polypeptide but lacking theheterologous polypeptide).

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces productionof HCV-specific CD4⁺ T cells and CD8⁺ T cells in the individual, wherethe number of HCV-specific CD4⁺ T cells and/or CD8⁺ T cells isincreased, such that the percent of total peripheral CD4⁺ and/or CD8⁺ Tcells that is HCV-specific is from 0.01% to 0.05%, from 0.05% to 0.10%,from 0.10% to 0.125%, from 0.125% to 0.25%, from 0.25% to from 0.50%, or0.5% to 10% (e.g., from 0.5% to 1%, from 1% to 2%, from 2% to 5%, orfrom 5% to 10%). The number of HCV-specific CD4⁺ T cells and CD8⁺ Tcells in a control individual (e.g., an individual not infected withHCV) not treated with the immunogenic composition would be undetectable.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces productionof HCV NS3-specific CD4⁺ T cells and/or CD8⁺ T cells in the individual,where the number of HCV NS3-specific CD4⁺ T cells and/or CD8⁺ T cells isincreased, such that the percent of the total peripheral blood T cells(i.e., the total number of CD4⁺ T cells+CD8⁺ T cells in the peripheralblood) that are HCV NS3-specific CD4⁺ T cells and CD8⁺ T cells is from0.01% to 10% (e.g., from 0.01% to 0.05%, from 0.05% to 0.1%, from 0.1%to 0.25%, from 0.25% to 0.5%, from 0.5% to 1%, from 1% to 2%, from 2% to5%, or from 5% to 10%). The number of HCV NS3-specific CD4⁺ T cells andCD8⁺ T cells in a control individual (e.g., an individual not infectedwith HCV) not treated with the immunogenic composition would beundetectable.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, increases the numberof HCV E1/E2-specific CD4⁺ T cells and CD8⁺ T cells in the individual byat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, compared to the number of HCV E1/E2-specific CD4⁺ T cells andCD8⁺ T cells in the individual induced by administration of a controlcomposition comprising the HCV E1/E2 heterodimer but lacking theheterologous polypeptide, or compared to the number of HCVE1/E2-specific CD4⁺ T cells and CD8⁺ T cells in the individual beforeadministration of the immunogenic composition.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces helper Tlymphocytes (e.g., CD4⁺ T cells) specific for HCV, where the number ofHCV-specific helper T lymphocytes induced is at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 7.5-fold, at least 10-fold, at least 20-fold, at least50-fold, or at least 100-fold, or more than 100-fold, higher than thenumber of HCV-specific helper T cells induced by administration of acontrol composition comprising the HCV E1/E2 heterodimer but lacking theheterologous polypeptide, or compared to the number of HCV-specific CD4⁺T cells in the individual before administration of the immunogeniccomposition.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces antibodyspecific for HCV, where the level of HCV-specific antibody induced is atleast at high as the level of HCV-specific antibody induced byadministration of a control composition comprising the HCV E1/E2heterodimer but lacking the heterologous polypeptide.

In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces antibodyspecific for HCV, where the level of HCV-specific antibody induced is atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, atleast 20-fold, at least 50-fold, or at least 100-fold, or more than100-fold, higher than the level of HCV-specific antibody induced byadministration of a control composition comprising the HCV E1/E2heterodimer but lacking the heterologous polypeptide, or compared thelevel of HCV-specific antibody in the individual before administrationof the immunogenic composition.

An immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response (e.g., acellular immune response) in the individual to one or more HCVgenotypes. In some cases, an immunogenic composition of the presentdisclosure, when administered to an individual in need thereof, inducesan immune response in the individual to HCV genotype 1. In some cases,an immunogenic composition of the present disclosure, when administeredto an individual in need thereof, induces an immune response in theindividual to HCV genotype 2. In some cases, an immunogenic compositionof the present disclosure when administered to an individual in needthereof, induces an immune response in the individual to HCV genotype 3.In some cases, an immunogenic composition of the present disclosure,when administered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1 and HCV genotype 3. In somecases, an immunogenic composition of the present disclosure, whenadministered to an individual in need thereof, induces an immuneresponse in the individual to HCV genotype 1, HCV genotype 2, and HCVgenotype 3. In some cases, an immunogenic composition of the presentdisclosure, when administered to an individual in need thereof, inducesan immune response in the individual to HCV genotype 1, HCV genotype 2,HCV genotype 3, and HCV genotype 7. In some cases, an immunogeniccomposition of the present disclosure, when administered to anindividual in need thereof, induces an immune response in the individualto HCV genotype 1, HCV genotype 2, HCV genotype 3, HCB genotype 4, HCVgenotype 5, HCV genotype 6, and HCV genotype 7.

HCV E1/E2 Heterodimers; HCV E2 Polypeptides; HCV E1 Polypeptides

HCV E1/E2 heterodimers suitable for use in an immunogenic composition ofthe present disclosure include HCV E1/E2 heterodimers comprisingwild-type HCV E1 polypeptides; HCV E1/E2 heterodimers comprisingwild-type HCV E2 polypeptides; HCV E1/E2 heterodimers comprising variantHCV E1 polypeptides; and HCV E1/E2 heterodimers comprising variant HCVE2 polypeptides. HCV E2 polypeptides suitable for use in an immunogeniccomposition of the present disclosure include wild-type E2 polypeptidesand variant E2 polypeptides. HCV E1 polypeptides suitable for use in animmunogenic composition of the present disclosure include wild-type E1polypeptides and variant E1 polypeptides.

E2 Polypeptides

An E2 polypeptide suitable for inclusion in an E1/E2 heterodimer forinclusion in an immunogenic composition of the present disclosure, orfor inclusion by itself in an immunogenic composition of the presentdisclosure, can have a length of from about 200 amino acids (aa) toabout 250 aa, from about 250 aa to about 275 aa, from about 275 aa toabout 300 aa, from about 300 aa to about 325 aa, from about 325 aa toabout 350 aa, or from about 350 aa to about 365 aa. In some cases, anHCV E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure is an HCV E2 ectodomain polypeptide. In somecases, an HCV E2 polypeptide suitable for inclusion in an immunogeniccomposition of the present disclosure is a full-length HCV E2polypeptide.

In FIG. 1A-AC, the amino acid sequence of E2 is amino acid 384 to aminoacid 746. In FIG. 2A-2B, the amino acid sequence of E2 is amino acid 384to amino acid 751. In FIG. 3A-3C, the amino acid sequence of E2 is aminoacid 385 to amino acid 754. In FIG. 4A-4B, the amino acid sequence of E2is amino acid 384 to amino acid 750. As used herein, an “E2 polypeptide”includes a precursor E2 protein, including the signal sequence; includesa mature E2 polypeptide which lacks this sequence; and includes an E2polypeptide with a heterologous signal sequence. An E2 polypeptide caninclude a C-terminal membrane anchor sequence which occurs atapproximately amino acid positions 715-730 and may extend as far asapproximately amino acid residue 746 (see, Lin et al., J. Virol. (1994)68:5063-5073).

In some cases, a E2 polypeptide suitable for inclusion in an immunogeniccomposition of the present disclosure lacks a portion of its C-terminalregion, e.g., from about amino acid 715 to the C-terminus; from aboutamino acid 625 to the C-terminus; from about amino acid 661 to theC-terminus; from about amino acid 655 to the C-terminus; from aboutamino acid 500 to the C-terminus, where the amino acid numbering is withreference to the numbering in FIG. 1A-1C. See, e.g., U.S. Pat. No.6,521,423.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.1A-1C, FIG. 2A-2C, FIG. 3A-3C, or FIG. 4A-4B. An E2 polypeptide suitablefor inclusion in an immunogenic composition of the present disclosurecan comprise an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, or at least about 75%, amino acid sequence identity to anamino acid sequence of an E2 polypeptide depicted in FIG. 1A-1C, FIG.2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.1A-1C. For example, an E2 polypeptide of genotype 1 can comprise anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 384-746 of an aminoacid sequence depicted in FIG. 1A-1C. For example, an E2 polypeptide ofgenotype 1A can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 384-746 of an amino acid sequence identified as 1A and depicted inFIG. 1A-1C. For example, an E2 polypeptide of genotype 1B can comprisean amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 384-746 of an aminoacid sequence identified as 1B and depicted in FIG. 1A-1C. For example,an E2 polypeptide of genotype 1C can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 384-746 of an amino acid sequenceidentified as 1C and depicted in FIG. 1A-1C.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.2A-2C. For example, an E2 polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 384-751 of an amino acid sequencedepicted in FIG. 2A-2C. For example, an E2 polypeptide of genotype 2Acan comprise an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 384-751of the “consensus” amino acid sequence depicted in FIG. 2A-2C. Forexample, an E2 polypeptide of genotype 2B can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 384-751 of the “consensus” aminoacid sequence depicted in FIG. 2A-2C.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an E2 polypeptide depicted in FIG.3A-3C. For example, an E2 polypeptide of genotype 3 can comprise anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 385-754 of an aminoacid sequence depicted in FIG. 3A-3C. For example, an E2 polypeptide ofgenotype 3A can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 385-754 of an amino acid sequence identified as 3A and depicted inFIG. 3A-3C. For example, an E2 polypeptide of genotype 3B can comprisean amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 385-754 of the aminoacid sequence identified as 3B and depicted in FIG. 3A-3C. For example,an E2 polypeptide of genotype 3K can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to amino acids 385-754 of the amino acid sequenceidentified as 3K and depicted in FIG. 3A-3C.

An E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the amino acid sequence of the E2 polypeptide depicted inFIG. 4A-4B. For example, an E2 polypeptide of genotype 7A can comprisean amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 384-750 of the aminoacid sequence depicted in FIG. 4A-4B.

E1 Polypeptides

An HCV E1 polypeptide suitable for inclusion in an E1/E2 heterodimer forinclusion in an immunogenic composition of the present disclosure, orfor inclusion by itself in an immunogenic composition of the presentdisclosure, can have a length of from about 100 amino acids (aa) toabout 150 aa, from about 150 aa to about 175 aa, from about 175 aa toabout 195 aa, from about 131 aa to about 175 aa, or from about 175 aa toabout 193 aa. In some cases, an HCV E1 polypeptide suitable forinclusion in an E1/E2 heterodimer present in an immunogenic compositionof the present disclosure is an HCV E1 ectodomain polypeptide. In somecases, an HCV E1 polypeptide suitable for inclusion in an E1/E2heterodimer present in an immunogenic composition of the presentdisclosure is a full-length HCV E1 polypeptide.

In FIG. 1A-1C, the amino acid sequence of E1 is amino acid 192 to aminoacid 383. In FIG. 2A-2C, the amino acid sequence of E1 is amino acid 192to amino acid 383. In FIG. 3A-3C, the amino acid sequence of E1 is aminoacid 192 to amino acid 384. In FIG. 4A-4B, the amino acid sequence of E1is amino acid 192 to amino acid 383. Amino acids at around 170 throughapproximately 191 serve as a signal sequence for E1. As used herein, “E1polypeptide” includes a precursor E1 protein, including the signalsequence; includes a mature E1 polypeptide which lacks this sequence;and includes an E1 polypeptide with a heterologous signal sequence. AnE1 polypeptide can include a C-terminal membrane anchor sequence whichoccurs at approximately amino acid positions 360-383 (see, e.g., WO96/04301). In some cases, a suitable E1 polypeptide lacks a C-terminalportion that includes a transmembrane region. For example, in somecases, a suitable E1 polypeptide lacks the C-terminal portion from aminoacid 330 to amino acid 384, or from amino acid 360 to amino acid 384. E1polypeptides can be an E1 polypeptide of any genotype, subtype orisolate of HCV. E1 polypeptides of genotype 1 and E1 polypeptides ofgenotype 3 are included in an E1/E2 heterodimer of the presentdisclosure.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 1A-1C, FIG.2A-2C, FIG. 3A-3C, or FIG. 4A-4B.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 1A-1C. Forexample, an E1 polypeptide of genotype 1A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of an amino acid sequenceidentified as 1A and depicted in FIG. 1A-1C. For example, an E1polypeptide of genotype 1B can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 192-383 of an amino acid sequence identified as1B and depicted in FIG. 1A-1C. For example, an E1 polypeptide ofgenotype 1C can comprise an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 192-383 of an amino acid sequence identified as 1C and depicted inFIG. 1A-1C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 2A-2C. Forexample, an E1 polypeptide of genotype 2A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of an amino acid sequenceidentified as 2A and depicted in FIG. 2A-2C. For example, an E1polypeptide of genotype 2B can comprise an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 192-383 of an amino acid sequence identified as2B and depicted in FIG. 2A-2C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity tothe consensus E1 polypeptide amino acid sequence depicted in FIG. 3A-3C.

An E1 polypeptide can comprise an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toan amino acid sequence of an E1 polypeptide depicted in FIG. 4A-4B. Forexample, an E1 polypeptide of genotype 7A can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to amino acids 192-383 of the amino acid sequencedepicted in FIGS. 4A-4B.

HCV E1 and E2 Polypeptides Comprising Amino Acids from a ProteolyticallyCleavable Linker

As described in more detail below, an HCV E1/E2 heterodimer can begenerated using a method that involves an HCV E1 or an HCV E2polypeptide comprising a heterologous proteolytically cleavable linker.Following enzymatic cleavage of the proteolytically cleavable linker,from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) heterologous amino acids canremain on the HCV E1 or E2 polypeptide. For example, from 1 to 6 (e.g.,1, 2, 3, 4, 5, or 6) heterologous amino acids can remain at theN-terminus of an HCV E2 polypeptide. As another example, from 1 to 6(e.g., 1, 2, 3, 4, 5, or 6) heterologous amino acids can remain at theC-terminus of an HCV E2 polypeptide. As another example, from 1 to 6(e.g., 1, 2, 3, 4, 5, or 6) heterologous amino acids can remain at theN-terminus of an HCV E1 polypeptide. As another example, from 1 to 6(e.g., 1, 2, 3, 4, 5, or 6) heterologous amino acids can remain at theC-terminus of an HCV E1 polypeptide.

In some cases, amino acids C-terminal to the proteolytic cleavage sitein a proteolytically cleavable linker are Gly-Pro, Ser, Gly, or Gly-Ser.Thus, in some cases, a modified HCV E1 polypeptide comprises, appendedto the N-terminus of an HCV E1 polypeptide: Gly-Pro, Ser, Gly, orGly-Ser. In other words, in some cases, a modified HCV E1 polypeptidecomprises, in order from N-terminus to C-terminus: a) Gly-Pro, Ser, Gly,or Gly-Ser; and b) an HCV E1 polypeptide.

In some cases, amino acids C-terminal to the proteolytic cleavage sitein a proteolytically cleavable linker are Gly-Pro, Ser, Gly, or Gly-Ser.Thus, in some cases, a modified HCV E2 polypeptide comprises, appendedto the N-terminus of an HCV E2 polypeptide: Gly-Pro, Ser, Gly, orGly-Ser. In other words, in some cases, a modified HCV E2 polypeptidecomprises, in order from N-terminus to C-terminus: a) Gly-Pro, Ser, Gly,or Gly-Ser; and b) an HCV E2 polypeptide.

In some cases, amino acids N-terminal to the proteolytic cleavage sitein a proteolytically cleavable linker are LEVLFQ (SEQ ID NO:76), ENLYYFQ(SEQ ID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR (SEQ ID NO:90), or DDDDK(SEQ ID NO:77). Thus, in some cases, a modified HCV E1 polypeptidecomprises, appended to the C-terminus of an HCV E1 polypeptide: LEVLFQ(SEQ ID NO:76), ENLYYFQ (SEQ ID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR(SEQ ID NO:90), or DDDDK (SEQ ID NO:77). In other words, in some cases,a modified HCV E1 polypeptide comprises, in order from N-terminus toC-terminus: a) an HCV E1 polypeptide; and b) LEVLFQ (SEQ ID NO:76),ENLYYFQ (SEQ ID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR (SEQ ID NO:90), orDDDDK (SEQ ID NO:77).

In some cases, amino acids N-terminal to the proteolytic cleavage sitein a proteolytically cleavable linker are LEVLFQ (SEQ ID NO:76), ENLYYFQ(SEQ ID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR (SEQ ID NO:90), or DDDDK(SEQ ID NO:77). Thus, in some cases, a modified HCV E2 polypeptidecomprises, appended to the C-terminus of an HCV E2 polypeptide: LEVLFQ(SEQ ID NO:76), ENLYYFQ (SEQ ID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR(SEQ ID NO:90), or DDDDK (SEQ ID NO:77). In other words, in some cases,a modified HCV E2 polypeptide comprises, in order from N-terminus toC-terminus: a) an HCV E2 polypeptide; and b) LEVLFQ (SEQ ID NO:76),ENLYYFQ (SEQ ID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR (SEQ ID NO:90), orDDDDK (SEQ ID NO:77).

In some cases, a flexible linker of from 1 to 10 amino acids isinterposed between the proteolytically cleavable linker and the HCV E1or E2 polypeptide. Flexible linkers are intrinsically disorderedflexible linker domains or loops that vary in length and can be rich inpolar uncharged amino acids. Flexible linkers include, e.g., glycinepolymers (G)_(n), glycine-serine polymers (including, for example,(GS)_(n), (GSGGS)_(n) (SEQ ID NO:56), (GGSGGS)_(n) (SEQ ID NO:57), and(GGGS)_(n) (SEQ ID NO:58), where n is an integer of at least one, e.g.,where n is 1, 2, 3, 4, 5, or 6); glycine-alanine polymers, such asGAGAGAGA and the like; and alanine-serine polymers, e.g., SASASASA andthe like. Exemplary linkers can comprise amino acid sequences including,but not limited to, GGSG (SEQ ID NO:59), GGSGG (SEQ ID NO:60), GSGSG(SEQ ID NO:61), GSGGG (SEQ ID NO:62), GGGSG (SEQ ID NO:63), GSSSG (SEQID NO:64), and the like.

For example, in some cases, a modified E1 polypeptide comprises, inorder from N-terminus to C-terminus: a) Gly-Pro, Ser, Gly, or Gly-Ser;b) a flexible linker of from 1 to 10 amino acids; and c) an HCV E1polypeptide.

As another example, in some cases, a modified E2 polypeptide comprises,in order from N-terminus to C-terminus: a) Gly-Pro, Ser, Gly, orGly-Ser; b) a flexible linker of from 1 to 10 amino acids; and c) an HCVE2 polypeptide.

As another in some cases, a modified E1 polypeptide comprises, fromN-terminus to C-terminus: a) an HCV E1 polypeptide; b) a flexible linkerof from 1 to 10 amino acids; and c) LEVLFQ (SEQ ID NO:76), ENLYYFQ (SEQID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR (SEQ ID NO:90), or DDDDK (SEQID NO:77).

As another in some cases, a modified E2 polypeptide comprises, fromN-terminus to C-terminus: a) an HCV E2 polypeptide; b) a flexible linkerof from 1 to 10 amino acids; and c) LEVLFQ (SEQ ID NO:76), ENLYYFQ (SEQID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR (SEQ ID NO:90), or DDDDK (SEQID NO:77).

E2 with N-Terminal Heterologous Amino Acids

In some cases, an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises a modifiedHCV E2 polypeptide with from 1 to 6 amino acids from the proteolyticallycleavable linker on the N-terminus of the E2 polypeptide. In some cases,an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises: a) an HCV E1polypeptide; and b) a modified E2 polypeptide comprising, in order fromN-terminus to C-terminus: i) from 1 to 6 heterologous amino acidswherein the from 1 to 6 heterologous amino acids are C-terminal to asite of proteolytic cleavage in a proteolytically cleavable linker; andii) an HCV E2 polypeptide.

Proteolytically cleavable linkers are described elsewhere herein.Following proteolytic cleavage of a precursor polypeptide, as describedherein, a modified E2 polypeptide is generated, which modified E2polypeptide comprises, at its N-terminus, amino acids C-terminal to theprotease cleavage site within the proteolytically cleavable linker.

For example, where the proteolytically cleavable linker comprises aPreScission cleavage site (LEVLFQGP; SEQ ID NO:65), where cleavageoccurs between the glutamine and the glycine, a modified E2 polypeptidepresent in a heterodimeric E1/E2 polypeptide suitable for inclusion inan immunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Gly-Pro; and b) an HCV E2 polypeptide.As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQS; SEQ ID NO:66), where cleavage occursbetween the glutamine and the serine, a modified E2 polypeptide presentin a heterodimeric E1/E2 polypeptide suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Ser; and b) an HCV E2 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQG; SEQ ID NO:67), where cleavage occursbetween the glutamine and the glycine, a modified E2 polypeptide presentin a heterodimeric E1/E2 polypeptide suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Gly; and b) an HCV E2 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa thrombin cleavage site (LVPRGS; SEQ ID NO:68), where cleavage occursbetween the arginine and the glycine, a modified E2 polypeptide presentin a heterodimeric E1/E2 polypeptide suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Gly-Ser; and an HCV E2 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa Factor Xa cleavage site (I(E/D)GRX, where X is any amino acid exceptarginine or proline; SEQ ID NO:69), where cleavage occurs between thearginine and the X, a modified E2 polypeptide present in a heterodimericE1/E2 polypeptide suitable for inclusion in an immunogenic compositionof the present disclosure comprises, in order from N-terminus toC-terminus: a) X (where X is any amino acid except arginine or proline);and an HCV E2 polypeptide.

Thus, for example, in some cases, an HCV E1/E2 heterodimer suitable forinclusion in an immunogenic composition of the present disclosurecomprises: a) an HCV E1 polypeptide; and b) a modified E2 polypeptidecomprising, in order from N-terminus to C-terminus: i) from 1 to 6heterologous amino acids, wherein the from 1 to 6 (e.g., 1, 2, 3, 4, 5,or 6) heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and ii) an HCV E2polypeptide. In some cases, the 1 to 6 heterologous amino acids areGly-Pro. In some cases, the 1 to 6 heterologous amino acids is Ser. Insome cases, the 1 to 6 heterologous amino acids is Gly. In some cases,the 1 to 6 heterologous amino acids are Gly-Ser.

As another example, in some cases, an HCV E1/E2 heterodimer suitable forinclusion in an immunogenic composition of the present disclosurecomprises: a) an HCV E2 polypeptide; and b) a modified E1 polypeptidecomprising, in order from N-terminus to C-terminus: i) from 1 to 6heterologous amino acids, wherein the from 1 to 6 (e.g., 1, 2, 3, 4, 5,or 6) heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and ii) an HCV E1polypeptide. In some cases, the 1 to 6 heterologous amino acids areGly-Pro. In some cases, the 1 to 6 heterologous amino acids is Ser. Insome cases, the 1 to 6 heterologous amino acids is Gly. In some cases,the 1 to 6 heterologous amino acids are Gly-Ser.

E1 with N-Terminal Heterologous Amino Acids

In some cases, an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises a modifiedHCV E1 polypeptide with from 1 to 6 amino acids from a proteolyticallycleavable linker on the N-terminus of the E1 polypeptide. In some cases,an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises: a) an HCV E2polypeptide; and b) a modified E1 polypeptide comprising, in order fromN-terminus to C-terminus: i) from 1 to 6 heterologous amino acidswherein the from 1 to 6 heterologous amino acids are C-terminal to asite of proteolytic cleavage in a proteolytically cleavable linker; andii) an HCV E1 polypeptide.

Proteolytically cleavable linkers are described elsewhere herein.Following proteolytic cleavage of a precursor polypeptide (e.g., aprecursor polypeptide comprising, in order from N-terminus toC-terminus: a) an Fc polypeptide or an HCV E2 polypeptide; b) aproteolytically cleavable linker; and c) an HCV E1 polypeptide), amodified E1 polypeptide is generated, which modified E1 polypeptidecomprises, at its N-terminus, amino acids C-terminal to the proteasecleavage site within the proteolytically cleavable linker.

For example, where the proteolytically cleavable linker comprises aPreScission cleavage site (LEVLFQGP; SEQ ID NO:65), where cleavageoccurs between the glutamine and the glycine, a modified E1 polypeptidepresent in an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) Gly-Pro; and b) an HCV E1 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQS; SEQ ID NO:66), where cleavage occursbetween the glutamine and the serine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) Ser; and b) an HCV E1 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQG; SEQ ID NO:67), where cleavage occursbetween the glutamine and the glycine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) Gly; and b) an HCV E1 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa thrombin cleavage site (LVPRGS; SEQ ID NO:68), where cleavage occursbetween the arginine and the glycine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) Gly-Ser; and an HCV E1 polypeptide.

As another example, where the proteolytically cleavable linker comprisesa Factor Xa cleavage site (I(E/D)GRX, where X is any amino acid exceptarginine or proline; SEQ ID NO:69), where cleavage occurs between thearginine and the X, a modified E1 polypeptide present in an HCV E1/E2heterodimer suitable for inclusion in an immunogenic composition of thepresent disclosure comprises, in order from N-terminus to C-terminus: a)X (where X is any amino acid except arginine or proline); and an HCV E1polypeptide.

E2 with C-Terminal Heterologous Amino Acids

In some cases, an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises a modifiedHCV E2 polypeptide with from 1 to 6 amino acids from a proteolyticallycleavable linker on the C-terminus of the E2 polypeptide. In some cases,an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises: a) an HCV E1polypeptide; and b) a modified E2 polypeptide comprising, in order fromN-terminus to C-terminus: i) an HCV E2 polypeptide; and ii) from 1 to 6heterologous amino acids wherein the from 1 to 6 heterologous aminoacids are N-terminal to a site of proteolytic cleavage in aproteolytically cleavable linker.

Proteolytically cleavable linkers are described elsewhere herein.Following proteolytic cleavage of a precursor polypeptide (e.g., aprecursor polypeptide comprising, in order from N-terminus toC-terminus: a) HCV E2 polypeptide; b) a proteolytically cleavablelinker; and c) an Fc polypeptide or an HCV E1 polypeptide), a modifiedE2 polypeptide is generated, which modified E2 polypeptide comprises, atits C-terminus, amino acids N-terminal to the protease cleavage sitewithin the proteolytically cleavable linker.

For example, where the proteolytically cleavable linker comprises aPreScission cleavage site (LEVLFQGP; SEQ ID NO:65), where cleavageoccurs between the glutamine and the glycine, a modified E2 polypeptidepresent in an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an HCV E2 polypeptide; and b) LEVLFQ(SEQ ID NO:76).

As another example, where the proteolytically cleavable linker comprisesan enterokinase cleavage site (DDDDK; SEQ ID NO:77), where cleavageoccurs C-terminal to the Lys, a modified E2 polypeptide present in anHCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E2 polypeptide; and b) DDDDK (SEQ IDNO:77).

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQG; SEQ ID NO:67), where cleavage occursbetween the glutamine and the glycine, a modified E2 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E2 polypeptide; and b) ENLYFQ.

As another example, where the proteolytically cleavable linker comprisesa thrombin cleavage site (LVPRGS; SEQ ID NO:68), where cleavage occursbetween the arginine and the glycine, a modified E2 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E2 polypeptide; and LVPR (SEQ IDNO:78).

As another example, where the proteolytically cleavable linker comprisesa Factor Xa cleavage site (I(E/D)GRX, where X is any amino acid exceptarginine or proline; SEQ ID NO:69), where cleavage occurs between thearginine and the X, a modified E2 polypeptide present in an HCV E1/E2heterodimer suitable for inclusion in an immunogenic composition of thepresent disclosure comprises, in order from N-terminus to C-terminus: a)an HCV E2 polypeptide; and I(E/D)GR (SEQ ID NO:90).

E1 with C-Terminal Heterologous Amino Acids

In some cases, an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises a modifiedHCV E1 polypeptide with from 1 to 6 amino acids from a proteolyticallycleavable linker on the C-terminus of the E1 polypeptide. In some cases,an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises: a) an HCV E2polypeptide; and b) a modified E1 polypeptide comprising, in order fromN-terminus to C-terminus: i) an HCV E1 polypeptide; and ii) from 1 to 6heterologous amino acids wherein the from 1 to 6 heterologous aminoacids are N-terminal to a site of proteolytic cleavage in aproteolytically cleavable linker.

Proteolytically cleavable linkers are described elsewhere herein.Following proteolytic cleavage of a precursor polypeptide (e.g., aprecursor polypeptide comprising, in order from N-terminus toC-terminus: a) HCV E1 polypeptide; b) a proteolytically cleavablelinker; and c) an Fc polypeptide or an HCV E2 polypeptide), a modifiedE1 polypeptide is generated, which modified E1 polypeptide comprises, atits C-terminus, amino acids N-terminal to the protease cleavage sitewithin the proteolytically cleavable linker.

For example, where the proteolytically cleavable linker comprises aPreScission cleavage site (LEVLFQGP; SEQ ID NO:65), where cleavageoccurs between the glutamine and the glycine, a modified E1 polypeptidepresent in an HCV E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure comprises, in orderfrom N-terminus to C-terminus: a) an HCV E1 polypeptide; and b) LEVLFQ(SEQ ID NO:76).

As another example, where the proteolytically cleavable linker comprisesan enterokinase cleavage site (DDDDK; SEQ ID NO:77), where cleavageoccurs C-terminal to the Lys, a modified E1 polypeptide present in anHCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E1 polypeptide; and b) DDDDK (SEQ IDNO:77).

As another example, where the proteolytically cleavable linker comprisesa TEV cleavage site (ENLYFQG; SEQ ID NO:67), where cleavage occursbetween the glutamine and the glycine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E1 polypeptide; and b) ENLYFQ.

As another example, where the proteolytically cleavable linker comprisesa thrombin cleavage site (LVPRGS; SEQ ID NO:68), where cleavage occursbetween the arginine and the glycine, a modified E1 polypeptide presentin an HCV E1/E2 heterodimer suitable for inclusion in an immunogeniccomposition of the present disclosure comprises, in order fromN-terminus to C-terminus: a) an HCV E1 polypeptide; and LVPR (SEQ IDNO:78).

As another example, where the proteolytically cleavable linker comprisesa Factor Xa cleavage site (I(E/D)GRX, where X is any amino acid exceptarginine or proline; SEQ ID NO:69), where cleavage occurs between thearginine and the X, a modified E1 polypeptide present in an HCV E1/E2heterodimer suitable for inclusion in an immunogenic composition of thepresent disclosure comprises, in order from N-terminus to C-terminus: a)an HCV E1 polypeptide; and I(E/D)GR (SEQ ID NO:90).

Additional Polypeptides

In any of the above-described embodiments, one or both of thepolypeptide chains of the E1/E2 heterodimer can include one or moreadditional polypeptides. For example, the E1 polypeptide, the E2polypeptide, or both the E1 and the E2 polypeptide, can include anaffinity tag. Suitable affinity tags include, e.g., immunoglobulin Fcpolypeptides, a poly(histidine) tag (e.g., His₆), a maltose bindingprotein (MBP), a glutathione-S-transferase (GST) polypeptide,calmodulin-binding peptide (CBP), Streptavidin-binding peptide (SBP),Strep-tag II, FLAG (e.g., DYKDDDDK (SEQ ID NO:91), hemagglutinin (HA)(e.g., YPYDVPDYA (SEQ ID NO:92), c-myc T7 ((e.g., EQKLISEEDL; SEQ IDNO:93), Glu-Glu, starch-binding domain (SBD), and Flag-Acidic-Target Tag(FATT), and the like.

In some cases, an E1/E2 heterodimer included in a composition of thepresent disclosure includes a variant E2 polypeptide. In some cases, theE1 polypeptide or the variant E2 polypeptide can include an Ig Fcpolypeptide at the C-terminus of the E1 polypeptide or the variant E2polypeptide. As another example, in some cases, the E1 polypeptide orthe variant E2 polypeptide can include an Ig Fc polypeptide at theN-terminus of the E1 polypeptide or the variant E2 polypeptide. Ig Fcpolypeptides are known in the art, and are described elsewhere herein.

Heterologous Polypeptides (T-Cell Epitope Polypeptides)

As noted above, in some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer, or an HCV E1polypeptide, or an HCV E2 polypeptide; and b) a heterologous polypeptidecomprising a T-cell epitope present in an HCV protein other than E1 andE2. In some cases, an immunogenic composition of the present disclosurecomprises an HCV E1/E2 heterodimer, and a heterologous polypeptidecomprising a T-cell epitope present in an HCV protein other than E1 andE2. In other cases, an immunogenic composition of the present disclosurecomprises an HCV E1 polypeptide, and a heterologous polypeptidecomprising a T-cell epitope present in an HCV protein other than E1 andE2. In other cases, an immunogenic composition of the present disclosurecomprises an HCV E2 polypeptide, and a heterologous polypeptidecomprising a T-cell epitope present in an HCV protein other than E1 andE2. Heterologous polypeptides suitable for inclusion in an immunogenicpolypeptide of the present disclosure comprise T cell epitopes that areconserved among different HCV genotypes leading to cross-reactivecellular immune responses. In some cases, the heterologous T-cellepitope polypeptide does not include a neotope; for example, in somecases, the heterologous T-cell epitope polypeptide does not include ajunction formed by amino acid sequences that do not naturally occuradjacent to one another in a naturally-occurring HCV polypeptide.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, or more than 10 (e.g., from 10 to 15, from 15 to 20,from 20 to 25, or from 25 to 30, or more than 30), T cell epitopes.T-cell epitopes are epitopes that, when presented with a majorhistocompatibility complex (MHC) (e.g., a human leukocyte antigen (HLA))Class I or MHC Class II molecule, are recognized and bound by a T-cellreceptor (TCR) present on a T cell surface. T-cell epitopes includeepitopes recognized by cytotoxic T cells (e.g., CD8⁺ T cells), andepitopes recognized by helper T cells (e.g., CD4⁺ T cells).

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the heterologous polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. A suitable source of T-cellepitopes includes non-toxic mutants of toxins, where the mutants arereferred to as “cross-reactive material (CRM).” Other examples of strongT helper epitopes are diphtheria toxoid, tetanus toxoid, meningococcalouter membrane protein, or mutant diphtheria protein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS3 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS3 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS3 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS3 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS3 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS3 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS3 CD4⁺ T cell epitope and at least one HCV-NS3 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS3 CD4⁺ T-cell epitopes and 2 or more HCV-NS3 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS3 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS3 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS2 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS2 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS2 T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS2 T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS2 CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS2 CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS2 CD4⁺ T cell epitope and at least one HCV-NS2 CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS2 CD4⁺ T-cell epitopes and 2 or more HCV-NS2 CD8⁺ T-cell epitopes.In some cases, the heterologous polypeptide comprises 2, 3, 4, or 5HCV-NS2 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS2 CD8⁺ T-cellepitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS4A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS4A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS4A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS4A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS4A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS4A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS4A CD4⁺ T cell epitope and at least one HCV-NS4A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS4A CD4⁺ T-cell epitopes and 2 or more HCV-NS4A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS4A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS4A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5A T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5A T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5A T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5A T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5A CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5A CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5A CD4⁺ T cell epitope and at least one HCV-NS5A CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5A CD4⁺ T-cell epitopes and 2 or more HCV-NS5A CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5A CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-NS5B T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-NS5B T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-NS5B T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-NS5B T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-NS5B CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-NS5B CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-NS5B CD4⁺ T cell epitope and at least one HCV-NS5B CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-NS5B CD4⁺ T-cell epitopes and 2 or more HCV-NS5B CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-NS5B CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5B CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-core T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-core T-cellepitopes. In some cases, the heterologous polypeptide comprises 4 ormore HCV-core T-cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD4⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD4⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesone or more HCV CD8⁺ T cell epitopes. In some cases, the heterologouspolypeptide comprises a single HCV-core CD8⁺ T-cell epitope. In somecases, the heterologous polypeptide comprises 2 or more HCV-core CD8⁺T-cell epitopes. In some cases, the heterologous polypeptide comprisesat least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the heterologous polypeptide comprises at leastone HCV-core CD4⁺ T cell epitope and at least one HCV-core CD8⁺ T cellepitope. In some cases, heterologous polypeptide comprises 2 or moreHCV-core CD4⁺ T-cell epitopes and 2 or more HCV-core CD8⁺ T-cellepitopes. In some cases, the heterologous polypeptide comprises 2, 3, 4,or 5 HCV-core CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-core CD8⁺T-cell epitopes.

In some cases, the heterologous polypeptide comprises a single T-cellepitope. In some cases, the heterologous polypeptide comprises a singleHCV-p7 T-cell epitope. In some cases, the heterologous polypeptidecomprises 2 or more T-cell epitopes. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 T-cell epitopes. In some cases,the heterologous polypeptide comprises 3 or more HCV-p7 T-cell epitopes.In some cases, the heterologous polypeptide comprises 4 or more HCV-p7T-cell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD4⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-core CD4⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises one or more HCV CD8⁺ Tcell epitopes. In some cases, the heterologous polypeptide comprises asingle HCV-p7 CD8⁺ T-cell epitope. In some cases, the heterologouspolypeptide comprises 2 or more HCV-p7 CD8⁺ T-cell epitopes. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope. In some cases,the heterologous polypeptide comprises at least one HCV-p7 CD4⁺ T cellepitope and at least one HCV-p7 CD8⁺ T cell epitope. In some cases,heterologous polypeptide comprises 2 or more HCV-p7 CD4⁺ T-cell epitopesand 2 or more HCV-p7 CD8⁺ T-cell epitopes. In some cases, theheterologous polypeptide comprises 2, 3, 4, or 5 HCV-p7 CD4⁺ T-cellepitopes and 2, 3, 4, or 5 HCV-p7 CD8⁺ T-cell epitopes.

In some cases, the heterologous polypeptide comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, or 63, of the T-cell epitopes set out in FIG. 9A-9B. In some cases,the heterologous polypeptide comprises from 1 to 3, from 3 to 5, from 5to 10, from 10 to 15, from 15 to 20, from 20 to 25, or from 25 to 30 ofthe T-cell epitopes set out in FIG. 9A-9B. For example, in some cases,the heterologous polypeptide comprises the T-cell epitopes designatedNS3-3, NS3-4, and NS3-11 in FIG. 9A-9B and FIG. 11A-11N. As anotherexample, in some cases, the heterologous polypeptide comprises theT-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG.9A-9B and FIG. 11A-11N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-3,NS3-4, NS3-5, and NS3-11 in FIG. 9A-9B and FIG. 11A-11N. As anotherexample, in some cases, the heterologous polypeptide comprises theT-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-11,NS3-12, and NS3-13 in FIG. 9A-9B and FIG. 11A-11N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 9A-9B and FIG. 11A-11N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated Core-1, Core-2, Core-3, Core-4, Core-5, Core-6,Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13, Core-14,Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, and Core-22 inFIG. 9A-9B and FIG. 11A-11N. As another example, in some cases, theheterologous polypeptide comprises the T-cell epitopes designated NS3-1,NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12,and NS3-13 in FIG. 9A-9B and FIG. 11A-11N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS2-1, NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8,NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 9A-9B and FIG. 11A-11N. As another example,in some cases, the heterologous polypeptide comprises the T-cellepitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7,NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1,NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, andNS4b-10 in FIG. 9A-9B and FIG. 11A-11N. As another example, in somecases, the heterologous polypeptide comprises the T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, and NS5b-2 in FIG. 9A-9B and FIG. 11A-11N. In somecases, the T-cell epitopes are contiguous. In some cases, any two T-cellepitopes are separated by linkers (e.g., a linker having a length offrom 1 amino acid to about 50 amino acids, e.g., from 1 amino acid to 5amino acids (aa), from 5 aa to 10 aa, from 10 aa to 15 aa, from 15 aa to20 aa, from 20 aa to 25 aa, from 25 aa to 30 aa, from 30 aa to 40 aa, orfrom 40 aa to 50 aa).

In some cases, the heterologous polypeptide comprises at least one HCVCD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope, whereepitopes are conserved among HCV genotypes 1 and 2. In some cases, theheterologous polypeptide comprises at least one HCV CD4⁺ T cell epitopeand at least one HCV CD8⁺ T cell epitope, where epitopes are conservedamong HCV genotypes 1 and 3. In some cases, the heterologous polypeptidecomprises at least one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺T cell epitope, where epitopes are conserved among HCV genotypes 1, 2,and 3. In some cases, the heterologous polypeptide comprises at leastone HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope,where epitopes are conserved among HCV genotypes 1, 2, 3, and 7. In somecases, the heterologous polypeptide comprises at least one HCV CD4⁺ Tcell epitope and at least one HCV CD8⁺ T cell epitope, where epitopesare conserved among HCV genotypes 1-7.

The heterologous polypeptide can have a length of from about 10 aminoacids to about 2000 amino acids; e.g., the heterologous polypeptide canhave a length of from 10 amino acids (aa) to 15 aa, from 15 aa to 20 aa,from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 450 aa to 500 aa, from 500 aa to 550 aa, from 550 aa to 600aa, from 600 aa to 650 aa, from 650 aa to 700 aa, from 700 aa to 750 aa,or from 750 aa to 800 aa. The heterologous polypeptide can have a lengthof from about 25 amino acids to about 2000 amino acids, e.g., from about25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa,from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa,from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to 400 aa,from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to 700 aa,from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to 1000 aa,from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aa to 1300aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500 aa to1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from 1800 aato 1900 aa, or from 1900 aa to 2000 aa. The heterologous polypeptide canhave a length of from about 25 amino acids to about 3000 amino acids,e.g., from about 25 amino acids (aa) to 50 aa, from 50 aa to 75 aa, from75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aato 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600 aa to700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa, from 900 aa to1000 aa, from 1000 aa to 1100 aa, from 1100 aa to 1200 aa, from 1200 aato 1300 aa, from 1300 aa to 1400 aa, from 1400 aa to 1500 aa, from 1500aa to 1600 aa, from 1600 aa to 1700 aa, from 1700 aa to 1800 aa, from1800 aa to 1900 aa, from 1900 aa to 2000 aa, from 2000 aa to 2250 aa,from 2250 aa to 2500 aa, from 2500 aa to 2750 aa, or from 2750 aa to3000 aa.

The heterologous polypeptide can have a length of from about 25 aminoacids to about 800 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from about 25 aminoacids to about 400 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, or from 350 aa to 400 aa. The heterologouspolypeptide can have a length of 25 amino acids (aa), 26 aa, 27 aa, 28aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37 aa, 38aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47 aa, 48aa, 49 aa, or 50 aa. The heterologous polypeptide can have a length offrom about 100 amino acids (aa) to 800 aa, e.g., from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The heterologous polypeptide can have a length of from 25 aa to 30 aa.The heterologous polypeptide can have a length of from 30 aa to 40 aa.The heterologous polypeptide can have a length of from 40 aa to 50 aa.The heterologous polypeptide can have a length of from 50 aa to 60 aa(e.g., 50 aa, 51 aa, 52, aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa,59 aa, or 60 aa). The heterologous polypeptide can have a length of from60 aa to 70 aa. The heterologous polypeptide can have a length of from65 aa to 75 aa (e.g., 65, 66, 67, 68, 69, 70, 71, 72, 7, 74, or 75 aa).The heterologous polypeptide can have a length of 70 aa. Theheterologous polypeptide can have a length of from 70 aa to 80 aa. Theheterologous polypeptide can have a length of from 80 aa to 90 aa. Theheterologous polypeptide can have a length of from 90 aa to 100 aa. Theheterologous polypeptide can have a length of from 100 aa to 105 aa(e.g., 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 aa). Theheterologous polypeptide can have a length of 100 aa. The heterologouspolypeptide can have a length of from 10 amino acids (aa) to 50 aa;e.g., from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 35 aa, from 35 aa to 40 aa, from 40aa to 45 aa, or from 45 aa to 50 aa. The heterologous polypeptide canhave a length of from 10 amino acids (aa) to 20 aa, e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 aa.

HCV NS3 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS3 polypeptide. Examples of T-cell epitopespresent in NS3 polypeptides are depicted in FIG. 11A-11N, FIG. 9B, andFIG. 10A-10B.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:94).AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:94) is referred to in FIG. 10Aas “TP29.” In some cases, the heterologous polypeptide comprises anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:94); and has a length of from25 aa to 35 aa (e.g., 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa,32 aa, 33 aa, 34 aa, or 35 aa). In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ IDNO:94); and has a length of 29 amino acids. Such a polypeptide caninclude NS3 T-cell epitopes designated NS3-3, NS3-4, and NS3-11 in FIG.9B and FIG. 11A-11N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95).AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95) isreferred to in FIG. 10A as “TP52.” In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95); andhas a length of from 45 amino acids to 60 amino acids (e.g., 45 aa, 46aa, 47 aa, 48 aa, 49 aa, 50 aa, 51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95); andhas a length of 52 amino acids. Such a polypeptide can include NS3T-cell epitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 9Band FIG. 11A-11N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96); and has a length of from 65 amino acids to 80 aminoacids (e.g., 65 aa, 66 aa, 67 aa, 68 aa, 69 aa, 70 aa, 71 aa, 72 aa, 73aa, 74 aa, 75 aa, 76 aa, 77 aa, 78 aa, 79 aa, or 80 aa).KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96) is referred to in FIG. 10A as “TP70.”

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96); and has a length of 70 amino acids. Such a polypeptidecan include NS3 T-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 9B and FIG. 11A-11N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97); and has alength of from 95 amino acids (aa) to 105 aa (e.g., 95 aa, 96 aa, 97 aa,98 aa, 99 aa, 100 aa, 101 aa, 102 aa, 103 aa, 104 aa, or 105 aa).VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97) is referred toin FIG. 10A as “TP100.”

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97); and has alength of 100 amino acids. Such a polypeptide can include NS3 T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 9B and FIG. 11A-11N.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and has a length of from 171 amino acids (aa) to 180 aa (e.g., 171 aa,172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa, 179 aa, or 180aa. MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89)is referred to in FIG. 10A as “TP171.”

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and has a length of 171 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:98); and has a length of from 190 aminoacids (aa) to 200 aa (e.g., 190 aa, 191 aa, 192 aa, 193 aa, 194 aa, 195aa, 196 aa, 197 aa, 198 aa, 199 aa, or 200 aa.

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:98); and has a length of 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:81); and hasa length of from 215 amino acids (aa) to 235 aa (e.g., 215 aa, 216 aa,217 11, 218 aa, 219 aa, 220 aa, 221 aa, 222 aa, 223 aa, 224 aa, 225 aa,226 aa, 227 aa, 228 aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa,or 235 aa). LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:81) isreferred to in FIG. 10A as “TP228.”

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:81);

-   -   and has a length of 228 amino acids. Such a polypeptide can        include NS3 T-cell epitopes designated NS3-1, NS3-2, NS3-3,        NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12, and        NS3-13 in FIG. 9B and FIG. 11A-11N.

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1265-1279 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1309-1323 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1401-1415 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1402-1412 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1429-1439 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1464 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1453-1467 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1577-1591 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1306-1314 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1387-1394 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 1amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1405-1413 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1450-1458 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1457-1465 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCV NS3T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1610-1618 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of any HCVgenotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS2 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS2 polypeptide. Examples of T-cell epitopespresent in NS2 polypeptides are depicted in FIG. 11A-11N, and FIG. 9A.

For example, the heterologous polypeptide can comprise an NS2 T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids955-974 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 975-994 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 985-1004 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1015-1034 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1035-1054 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 924-933 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 961-970 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the heterologous polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 989-997 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 50 aa (e.g., from 10 aa to 25 aa, or from 25 aa to 50 aa) ofamino acids 955-1004 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa, orfrom 25 aa to 50 aa. In some cases, the heterologous polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 955-1004of the amino acid sequence designated “Consensus” in FIG. 12A-12L, or acorresponding HCV NS2 amino acid sequence of any HCV genotype; and has alength of about 50 amino acids.

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 553 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from 300 aato 400 aa, from 400 aa to 500 aa, or from 500 aa to 553 aa) of aminoacids 917-1469 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 and NS3 amino acid sequence of anyHCV genotype; and has a length of from 10 amino acids (aa) to 25 aa,from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 200 aa, from200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, or from500 aa to 553 aa. In some cases, the heterologous polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 917-1469 of the aminoacid sequence designated “Consensus” in FIG. 12A-12L, or a correspondingHCV NS2 and NS3 amino acid sequence of any HCV genotype; and has alength of about 553 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 0%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99).LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99) isreferred to in FIG. 10A as “TP50.” In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99); andhas a length of from 50 amino acids to 60 amino acids (e.g., 50 aa, 51aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa).In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99); andhas a length of 50 amino acids. Such a polypeptide can include NS2T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG.9A and FIG. 11A-11N.

HCV NS4A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS4A polypeptide. Examples of T-cell epitopespresent in NS4A polypeptides are depicted in FIG. 11A-11N and FIG. 9B.

The heterologous polypeptide can comprise an NS4A T cell epitopecomprising an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids1683-1692 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS4A amino acid sequence of any HCVgenotype; and the NS4A T-cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

HCV NS4B T-Cell Epitopes

In some cases, the heterologous polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS4B polypeptide. Examples of T-cell epitopespresent in NS4B polypeptides are depicted in FIG. 11A-11N and FIG. 9B.

As one example, the heterologous polypeptide can comprise an NS4B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids1790-1801 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 12 aminoacids (aa) to 20 amino acids (e.g., 12 aa, 13 aa, 14 aa, 15 aa, 16 aa,17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1792-1802 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 11 aminoacids (aa) to 20 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15 aa,16 aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1898-1905 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 8 aminoacids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1921-1935 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1922-1941 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1928-1947 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1868-1876 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1927-1942 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 16 aminoacids (aa) to 20 amino acids (e.g., 16 aa, 17 aa, 18 aa, 19 aa, or 20aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1932-1940 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an NS4B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1948-1962 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

HCV NS5A T-Cell Epitopes

In some cases, the heterologous polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS5A polypeptide. Examples of T-cell epitopespresent in NS5A polypeptides are depicted in FIG. 11A-11N and FIG. 9B.

As one example, the heterologous polypeptide can comprise an NS5A T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2218-2232 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the heterologous polypeptide can comprise an NS5A Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2309-2317 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV NS5B T-cell epitopes

In some cases, the heterologous polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV NS5B polypeptide. Examples of T-cell epitopespresent in NS5B polypeptides are depicted in FIG. 11A-11N and FIG. 9B.

As one example, the heterologous polypeptide can comprise an NS5B T cellepitope comprising an amino acid sequence having at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 60%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to amino acids2847-2851 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 5 aminoacids (aa) to 10 amino acids (e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa).

As another example, the heterologous polypeptide can comprise an NS5B Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2602-2610 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

HCV Core T-Cell Epitopes

In some cases, the heterologous polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV core polypeptide. Examples of T-cell epitopespresent in HCV Core polypeptides are depicted in FIG. 11A-11N and FIG.9A.

As one example, the heterologous polypeptide can comprise an HCV core Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1-20 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 11-30 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 21-40 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 39-63 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 23amino acids (aa) to 28 amino acids (e.g., 23 aa, 24 aa, 25 aa, 26 aa, 27aa, or 28 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 47-70 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 24amino acids (aa) to 29 amino acids (e.g., 24 aa, 25 aa, 26 aa, 27 aa, 28aa, or 29 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 61-80 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 71-90 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 81-100 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 91-110 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 101-115 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 111-130 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 125-139 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-150 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 151-170 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 161-180 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 35-44 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 43-51 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 51-59 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 129-137 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 131-140 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 150-158 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 154-162 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 168-176 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 177-187 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 11amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, or 16 aa).

As another example, the heterologous polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 178-187 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 10amino acids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 10 amino acids(aa) to 191 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa, from 50aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, or from 150 aato 191aa) of amino acids 1-191 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and has a length of from 10 amino acids(aa) to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, or from 100 aato 150 aa, or from 150 aa to 191 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to amino acids1-191 of the amino acid sequence designated “Consensus” in FIG. 12A-12L,or a corresponding HCV core amino acid sequence of any HCV genotype; andhas a length of about 191 amino acids.

The heterologous polypeptide can comprise an amino acid sequence havingat least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and has a length of from 171 amino acids (aa) to 180 aa (e.g., 171 aa,172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa, 179 aa, or 180aa. In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and has a length of 171 amino acids. Such a polypeptide can include coreT-cell epitopes designated Core-1, Core-2, Core-3, Core-4, Core-5,Core-6, Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13,Core-14, Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, Core-22in FIG. 9A and FIG. 11A-11N.

HCV p7 T-Cell Epitopes

In some cases, the heterologous polypeptide present in an immunogeniccomposition of the present disclosure includes one or more T-cellepitopes present in an HCV p7 polypeptide. Examples of T-cell epitopespresent in HCV p7 polypeptides are depicted in FIG. 11A-11N or FIG. 9A.

As another example, the heterologous polypeptide can comprise an HCV p7T cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 803-811 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV p7 amino acid sequence of any HCVgenotype; and the HCV p7 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

Heterologous Polypeptides Including HCV T-Cell Epitopes from More thanOne HCV Polypeptide Other than E1 and E2

As noted above, a heterologous polypeptide can include T-cell epitopesfrom more than one HCV polypeptide other than E1 and E2.

As one example, a heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100); and has a length of from 550amino acids (aa) to 560 aa (e.g., 550 aa, 551 aa, 552 aa, 553 aa, 554aa, 555 aa, 556 aa, 557 aa, 558 aa, 559 aa, or 560 aa).

QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100) is referred to in FIG. 10A-10Bas “TP553.”

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100); and has a length of 553 aminoacids. Such a polypeptide can include T-cell epitopes designated NS2-1,NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8, NS3-1, NS3-2, NS3-3,NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 inFIG. 9A-9B and FIG. 11A-11N. This polypeptide is also referred to as“TP553” (FIG. 12A-12D). In order to prevent self cleavage of the TP553polypeptide (amino acids 917-1469) (FIG. 11E-11G) at the NS2-NS3junction that is mediated by the catalytic domain of the NS2 protease(amino acids 917-1040), the histidine at position 966 (H966), a criticalresidue for NS2 protease activity, is mutated to alanine (H966A) (FIG.11E). See, e.g., Grakoui, A. et al. A second hepatitis C virus-encodedproteinase. Proc. Natl Acad. Sci. USA 90, 10583-10587 (1993); Hijikata,M. et al. Two distinct proteinase activities required for the processingof a putative nonstructural precursor protein of hepatitis C virus. J.Virol. 67, 4665-4675 (1993); and Lorenz. I C. Structure of the catalyticdomain of the hepatitis C virus NS2-3 protease. Nature. August 17;442(7104):831-5 (2006).

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aato 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa) the followingamino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and has a length of from 778 amino acids (aa) to 790 aa(e.g., 778 aa, 779 aa, 780 aa, 781 aa, 782 aa, 783 aa, 784 aa, 785 aa,786 aa, 787 aa, 788 aa, or 790 aa).

LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPG (SEQ ID NO:101) is referred to in FIG. 10B as “TP778.”

In some cases, the heterologous polypeptide comprises an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from 25 amino acids(aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa, from 75aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from 200 aato 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350 aa to400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aa to 550aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to 700 aa,from 700 aa to 750 aa, or from 750 aa to 778 aa) of the following aminoacid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and has a length of from 25 amino acids (aa) to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600aa to 700 aa, or from 700 aa to 778 aa. In some cases, the heterologouspolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and has a length of 778 amino acids. Such a polypeptidecan include T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4,NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13,NS2-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7,NS4b-8, NS4b-9, and NS4b-10 in FIG. 9B and FIG. 11A-11N.

As another example, the heterologous polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 1985 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 500 aa, from 500 aa to 750 aa, from 750aa to 1000 aa, from 1000 aa to 1500 aa, or from 1500 aa to 1985 aa) ofthe following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:102); this polypeptide isalso referred to as “TP1985” and is depicted in FIG. 10C.

In some cases, the heterologous polypeptide can comprise an amino acidsequence having at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:102); and has a length of1985 amino acids. Such a polypeptide can include T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, NS5b-2 in FIG. 9A-9B and FIG. 11A-11N.

Additional T-Cell Epitopes

As discussed above, an immunogenic composition of the present disclosureincludes: a) an HCV E1/E2 heterodimer; and b) a heterologous polypeptidethat comprises one or more T-cell epitopes (e.g., one or more T cellepitopes present in an HCV polypeptide other than an HCV E1 polypeptideor an HCV E2 polypeptide). The one or more T-cell epitopes can includeone or more T-cell epitopes present in: a) an HCV NS3 polypeptide; b) anHCV NS2 polypeptide; c) an HCV NS4A polypeptide; d) an HCV NS4Bpolypeptide; e) an HCV NS5A polypeptide; f) an HCV NS5B polypeptide; g)an HCV core polypeptide; or h) an HCV p7 polypeptide. In some cases, theone or more T-cell epitopes are T-cell epitopes present in an HCV NS3polypeptide. In some cases, the heterologous polypeptide furthercomprises one or more T cell epitopes present in: a) cholera toxin ortoxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheria toxin ortoxoid; and/or d) a meningococcal outer membrane protein.

Thus, in some cases, an immunogenic composition of the presentdisclosure includes: a) an HCV E1/E2 heterodimer; and b) a heterologouspolypeptide that comprises one or more T-cell epitopes, where the one ormore T-cell epitopes are T-cell epitopes present in: i) one or more ofan HCV NS3 polypeptide, an HCV NS2 polypeptide, an HCV NS4A polypeptide,an HCV NS4B polypeptide, an HCV NS5A polypeptide, an HCV NS5Bpolypeptide, an HCV core polypeptide, and an HCV p7 polypeptide; and ii)one or more of cholera toxin or toxoid, tetanus toxin or toxoid,diphtheria toxin or toxoid, and a meningococcal outer membrane protein.

A T helper tetanus toxin epitope or other bacterial T-cell epitope couldbe fused (e.g., by recombinant expression) or chemically conjugated tothe heterologous polypeptide, or can be unconjugated (e.g., provided asa separate polypeptide), to further enhance both T and B cell responsesto both the T-cell epitopes present in the heterologous polypeptide andin the E1/E2 polypeptides. Alternatively, the whole or part of thedetoxified toxin (“toxoid”) can be used, wherein specific amino acids ofthe toxins are mutated to render the toxins inactive, thereby generatingtoxoids. Methods of generating toxoids are well known in the art.Examples of bacterial epitopes include the use of diphtheria toxoid,meningococcal outer membrane protein, or mutant diphtheria proteinCRM197 (see, e.g.: http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, a suitable tetanus toxoid polypeptide comprises the aminoacid sequence QYIKANSKFIGIFE (SEQ ID NO:103). In some cases, a suitabletetanus toxoid polypeptide comprises the amino acid sequenceQYIKANSKFIGITE (SEQ ID NO:104).

In some cases, a heterologous polypeptide can comprise cholera toxin (ortoxoid) epitope. In some cases, a suitable heterologous polypeptidecomprising a cholera toxoid epitope comprises a fragment of choleratoxin-B subunit (CT-B), e.g., a fragment of from 5 amino acids to 25amino acids, or from 25 amino acids to 50 amino acids, of the followingamino acid sequence: MIKLKFGVFF TVLLSSAYAH GTPQNITDLC AEYHNTQIHTLNDKIFSYTE SLAGKREMAI ITFKNGATFQ VEVPGSQHID SQKKAIERMK DTLRIAYLTEAKVEKLCVWN NKTPHAIAAI SMAN (SEQ ID NO:105).

In some cases, a heterologous polypeptide can comprise a tetanus toxin(or toxoid) T-cell epitope. In some cases, a suitable heterologouspolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: ILMQYIKANSKFIGI (SEQ ID NO:106); and has a lengthof from 15 amino acids to 20 amino acids. In some cases, a suitableheterologous polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: VNNESSE (SEQ ID NO:107). In somecases, a suitable heterologous polypeptide comprising a tetanus toxinT-cell epitope comprises the amino acid sequence: PGINGKAIHLVNNESSE (SEQID NO:108). In some cases, a suitable heterologous polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: PNRDIL (SEQ ID NO:109). In some cases, a suitable heterologouspolypeptide comprising a tetanus toxin T-cell epitope comprises theamino acid sequence: FIGITEL (SEQ ID NO:110). In some cases, a suitabletetanus toxin T-cell epitope comprises the amino acid sequence: SYFPSV(SEQ ID NO:111). In some cases, a suitable heterologous polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: NSVDDALINSTKIYSYFPSV (SEQ ID NO:112). In some cases, asuitable heterologous polypeptide comprising a tetanus toxin T-cellepitope comprises the amino acid sequence: IDKISDVSTIVPYIGPALNI (SEQ IDNO:113).

In some cases, a heterologous polypeptide can comprise a diphtheriatoxin T-cell epitope In some cases, a suitable heterologous polypeptidecomprising a diphtheria toxin T-cell epitope comprises the amino acidsequence: QSIALSSLMVAQAIP (SEQ ID NO:114); and has a length of from 15amino acids to 20 amino acids. In some cases, a suitable heterologouspolypeptide comprising a diphtheria toxin T-cell epitope comprises theamino acid sequence: PVFAGANYAAWAVNVAQVI (SEQ ID NO:115). In some cases,a suitable heterologous polypeptide comprising a diphtheria toxin T-cellepitope comprises the amino acid sequence: VHHNTEEIVAQSIALSSLMV (SEQ IDNO:116). In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QSIALSSLMVAQAIPLVGEL (SEQ ID NO:117). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: VDIGFAAYNFVESIINLFQV (SEQ ID NO:118).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QGESGHDIKITAENTPLPIA (SEQ ID NO:119). In some cases, a suitableheterologous polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: GVLLPTIPGKLDVNKSKTHI (SEQ ID NO:120).In some cases, a suitable heterologous polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence ofCRM197 (see, e.g., Giannini et al. (1984) Nucl. Acids. Res. 12:4063).

The amino acid sequence of CRM197 is as follows:

(SEQ ID NO: 121) laddvvdssksfvmenfssyhgtkpgyvdsiqkgiqkpksgtqgnydddwkefystdnkydaagysvdnenplsgkaggvvkvtypgltkvlalkvdnaetikkelglslteplmeqvgteefikrfgdgasrvvlslpfaegsssveyinnweqakalsveleinfetrgkrgqdamyeymaqacagnrvrrsvgsslscinldwdvirdktktkieslkehgpiknkmsespnktvseekakqyleefhqtalehpelselktvtgtnpvfaganyaawavnvaqvidsetadnlekttaalsilpgigsvmgiadgavhhnteeivaqsialsslmvaqaiplvgelvdigfaaynfvesiinlfqvvhnsynrpayspghktqpflhdgyavswntvedsiirtgfqgesghdikitaentplpiagvllptipgkldvnkskthisvngrkirmrcraidgdvtfcrpkspvyvgngvhanlhvafhrsssekihsneissdsigvlgyqktvdhtkvnsklslffeiks.

In some cases, a heterologous polypeptide can comprise a tetanus toxinT-cell epitope and a diphtheria toxin T-cell epitope. In some of thesecases, the heterologous polypeptide can comprise the amino acidsequence: IMQYIKANSKFIGIQSIALSSLMVAQ (SEQ ID NO:122); and can have alength of from 26 amino acids to 30 amino acids.

Mixtures of Heterologous Polypeptides (T-Cell Epitope Polypeptides)

In some cases, an immunogenic composition of the present disclosurecomprises two or more different heterologous polypeptides comprising aT-cell epitope present in an HCV protein other than E1 and E2 (e.g., amixture of two or more different heterologous polypeptides comprising aT-cell epitope present in an HCV protein other than E1 and E2).

For example, in some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimeric polypeptide; b) twoor more different heterologous polypeptides comprising a T-cell epitopepresent in an HCV protein other than E1 and E2; and c) apharmaceutically acceptable excipient. In some cases, an immunogeniccomposition of the present disclosure comprises: a) an HCV E2polypeptide; b) two or more different heterologous polypeptidescomprising a T-cell epitope present in an HCV protein other than E1 andE2; and c) a pharmaceutically acceptable excipient.

For example, the two or more different heterologous polypeptides caninclude: i) a first heterologous polypeptide comprising an amino acidsequence having at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or 100%, amino acid sequence identity to TP29, and having alength of from 29 amino acids to 35 amino acids; and ii) a secondheterologous polypeptide comprising an amino acid sequence having atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP50, and having a length of from50 amino acids to 55 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP29, andhaving a length of from 29 amino acids to 35 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP52, and having a length of from52 amino acids to 60 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP29, andhaving a length of from 29 amino acids to 35 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP70, and having a length of from70 amino acids to 75 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP29, andhaving a length of from 29 amino acids to 35 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP100, and having a length of from100 amino acids to 110 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP29, andhaving a length of from 29 amino acids to 35 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP171, and having a length of from171 amino acids to 180 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP29, andhaving a length of from 29 amino acids to 35 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP228, and having a length of from228 amino acids to 235 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP29, andhaving a length of from 29 amino acids to 35 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP553, and having a length of from553 amino acids to 565 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP29, andhaving a length of from 29 amino acids to 35 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP778, and having a length of from778 amino acids to 785 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP50, andhaving a length of from 50 amino acids to 55 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP52 and having a length of from52 amino acids to 60 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP50, andhaving a length of from 50 amino acids to 55 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP70 and having a length of from70 amino acids to 80 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP50, andhaving a length of from 50 amino acids to 55 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP100 and having a length of from100 amino acids to 110 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP50, andhaving a length of from 50 amino acids to 55 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP171 and having a length of from171 amino acids to 180 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP50, andhaving a length of from 50 amino acids to 55 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP228 and having a length of from228 amino acids to 240 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP50, andhaving a length of from 50 amino acids to 55 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP553 and having a length of from553 amino acids to 570 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP50, andhaving a length of from 50 amino acids to 55 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP778 and having a length of from778 amino acids to 790 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP52, andhaving a length of from 52 amino acids to 60 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP70 and having a length of from70 amino acids to 80 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP52, andhaving a length of from 52 amino acids to 60 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP100 and having a length of from100 amino acids to 110 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP52, andhaving a length of from 52 amino acids to 60 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP171 and having a length of from171 amino acids to 180 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP52, andhaving a length of from 52 amino acids to 60 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP228 and having a length of from228 amino acids to 240 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP52, andhaving a length of from 52 amino acids to 60 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP553 and having a length of from553 amino acids to 570 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP52, andhaving a length of from 52 amino acids to 60 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP778 and having a length of from778 amino acids to 790 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP70, andhaving a length of from 70 amino acids to 80 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP100 and having a length of from100 amino acids to 110 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP70, andhaving a length of from 70 amino acids to 80 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP171 and having a length of from171 amino acids to 190 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP70, andhaving a length of from 70 amino acids to 80 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP228 and having a length of from228 amino acids to 240 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP70, andhaving a length of from 70 amino acids to 80 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP553 and having a length of from553 amino acids to 570 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP70, andhaving a length of from 70 amino acids to 80 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP778 and having a length of from778 amino acids to 790 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP29, andhaving a length of from 29 amino acids to 35 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP50, andhaving a length of from 50 amino acids to 55 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP52, andhaving a length of from 52 amino acids to 60 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP70, andhaving a length of from 70 amino acids to 80 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP100, andhaving a length of from 100 amino acids to 115 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP171, andhaving a length of from 171 amino acids to 180 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP228, andhaving a length of from 228 amino acids to 235 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP553, andhaving a length of from 553 amino acids to 560 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

As another example, the two or more different heterologous polypeptidescan include: i) a first heterologous polypeptide comprising an aminoacid sequence having at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, or 100%, amino acid sequence identity to TP778, andhaving a length of from 778 amino acids to 790 amino acids; and ii) asecond heterologous polypeptide comprising an amino acid sequence havingat least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100%, amino acid sequence identity to TP1985 and having a length of from1985 amino acids to 2000 amino acids.

Pharmaceutically Acceptable Excipients

The present disclosure provides an immunogenic composition comprising:a) an HCV heterodimeric polypeptide comprising: i) an HCV E1polypeptide; and ii) an HCV E2 polypeptide; b) a heterologouspolypeptide comprising a T-cell epitope present in an HCV protein otherthan E1 and E2; and c) a pharmaceutically acceptable carrier. Thepresent disclosure provides an immunogenic composition comprising: a) anHCV E2 polypeptide; b) a heterologous polypeptide comprising a T-cellepitope present in an HCV protein other than E1 and E2; and c) apharmaceutically acceptable carrier. The present disclosure provides animmunogenic composition comprising: a) an HCV E1 polypeptide; b) aheterologous polypeptide comprising a T-cell epitope present in an HCVprotein other than E1 and E2; and c) a pharmaceutically acceptablecarrier.

In some cases, where an immunogenic composition of the presentdisclosure includes an HCV E1 polypeptide and an HCV E2 polypeptide, theratio of HCV E2 polypeptide to HCV E1 polypeptide is in a range of fromabout 2:1 to 1:1, e.g., from about 2:1 to 1.5:1, or from 1.5:1 to 1:1.In some cases, where an immunogenic composition of the presentdisclosure includes an HCV E1 polypeptide and a HCV E2 polypeptide, themolar ratio of HCV E2 polypeptide to HCV E1 polypeptide is in a range offrom about 1:1 to 1.5:1, from 1.5:1 to 2:1, from 2:1 to 3:1, from 3:1 to4:1, from 4:1 to 6:1, or from 6:1 to 8:1.

HCV E1 polypeptides, HCV E2 polypeptides, and heterologous polypeptidescan be formulated with a pharmaceutically acceptable excipient(s) togenerate an immunogenic composition of the present disclosure. A widevariety of pharmaceutically acceptable excipients is known in the artand need not be discussed in detail herein. Pharmaceutically acceptableexcipients have been amply described in a variety of publications,including, for example, A. Gennaro (2000) “Remington: The Science andPractice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins;Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Anselet al., eds 7th ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer.Pharmaceutical Assoc.

In some embodiments, an HCV E1 polypeptide, an HCV E2 polypeptide (e.g.,as an HCV E1/E2 heterodimer), and a heterologous polypeptide areformulated in an aqueous buffer. Suitable aqueous buffers include, butare not limited to, acetate, succinate, citrate, and phosphate buffersvarying in strengths from about 5 mM to about 100 mM. In someembodiments, the aqueous buffer includes reagents that provide for anisotonic solution. Such reagents include, but are not limited to, sodiumchloride; and sugars e.g., mannitol, dextrose, sucrose, and the like. Insome embodiments, the aqueous buffer further includes a non-ionicsurfactant such as polysorbate 20 (TWEEN®20) or polysorbate 80(TWEEN®80). For example, a formulation of an HCV E1 polypeptide, an HCVE2 polypeptide (e.g., as an HCV E1/E2 heterodimer), and a heterologouspolypeptide in an aqueous buffer can include, e.g., from about 0.01% toabout 0.05% polysorbate-20 (TWEEN®20) non-ionic detergent. Optionallythe formulations may further include a preservative. Suitablepreservatives include, but are not limited to, a benzyl alcohol, phenol,chlorobutanol, benzalkonium chloride, and the like. In many cases, theformulation is stored at about 4° C. Formulations may also belyophilized, in which case they generally include cryoprotectants suchas sucrose, trehalose, lactose, maltose, mannitol, and the like.Lyophilized formulations can be stored over extended periods of time,even at ambient temperatures. In some cases, the aqueous buffer furtherincludes a non-ionic surfactant. In some cases, the aqueous bufferincludes the non-ionic surfactant Triton™X-100, e.g., 0.1% Triton™X-100.

An HCV E1 polypeptide, an HCV E2 polypeptide (e.g., as an HCV E1/E2heterodimer), and a heterologous polypeptide can be formulated intopreparations for injection by dissolving, suspending or emulsifying themin an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives.

An immunogenic composition of the present disclosure can include, e.g.,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like.

The concentration of an HCV E1 polypeptide, an HCV E2 polypeptide (e.g.,as an HCV E1/E2 heterodimer), and a heterologous polypeptide in aformulation can vary widely (e.g., from less than about 0.1% to at leastabout 2%, to as much as 20% to 50% or more by weight) and can beselected primarily based on fluid volumes, viscosities, andpatient-based factors in accordance with the particular mode ofadministration selected and the patient's needs.

An immunogenic composition of the present disclosure can be provided inthe form of a solution, suspension, tablet, pill, capsule, powder, gel,cream, lotion, ointment, aerosol or the like. It is recognized that oraladministration can require protection of the compositions fromdigestion. This is typically accomplished either by association of thecomposition with an agent that renders it resistant to acidic andenzymatic hydrolysis or by packaging the composition in an appropriatelyresistant carrier. Means of protecting from digestion are well known inthe art.

An immunogenic composition of the present disclosure can also beprovided so as to enhance serum half-life of the polypeptides (an HCV E1polypeptide, an HCV E2 polypeptide (e.g., as an HCV E1/E2 heterodimer),and a heterologous polypeptide) following administration. For example,where an isolated HCV E1 polypeptide, an HCV E2 polypeptide (e.g., as anHCV E1/E2 heterodimer), and a heterologous polypeptide are formulatedfor injection, the polypeptides may be provided in a liposomeformulation, prepared as a colloid, or other conventional techniques forextending serum half-life. A variety of methods are available forpreparing liposomes, as described in, e.g., Szoka et al., Ann Rev.Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and4,837,028. The preparations may also be provided in controlled releaseor slow-release forms.

Adjuvant

An immunogenic composition of the present disclosure can include anadjuvant. Examples of known suitable adjuvants that can be used inhumans include, but are not necessarily limited to, alum, aluminumphosphate, aluminum hydroxide, MF59™ (4.3% w/v squalene, 0.5% w/v Tween80™ (polysorbate 80), 0.5% w/v Sorbitan trioleate (Span® 85)),CpG-containing nucleic acid (where the cytosine is unmethylated), QS21,monophosphoryl lipid A (MPL), 3-Q-desacyl-4′-monophosphoryl lipid A(3DMPL), extracts from Aquilla, immune-stimulating complexes (ISCOMS;complexes of cholesterol, phospholipids, and Quillaia saponins), LT/CTmutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, saponinvaccine adjuvant (Quil A®), interleukins, and the like. For experimentalanimals, one can use Freund's incomplete adjuvant, or Freund's completeadjuvant. Also suitable for use areN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI™, which contains threecomponents extracted from bacteria: monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene Tween80™ (polysorbate 80) emulsion. The effectiveness of an adjuvant may bedetermined by one or more of measuring the amount of antibodies directedagainst the immunogenic antigen or antigenic epitope thereof, measuringa cytotoxic T lymphocyte response to the antigen, and measuring a helperT cell response to the antigen.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59™ (see, e.g., WO 90/14837), containing 5% Squalene, 0.5%Tween 80™ (polysorbate 80), and 0.5% Sorbitan trioleate (Span® 85)(optionally containing MTP-PE) formulated into submicron particles usinga microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80™(polysorbate 80), 5% pluronic-blocked polymer L121, and thr-MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) RIBI™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80™ (polysorbate 80), and one or more bacterial cell wall componentssuch as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cellwall skeleton (CWS), e.g., MPL+CWS (Detox™); (2) saponin adjuvants, suchas QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.; a purifiedextract of Quillaja saponaria) may be used or particles generatedtherefrom such as ISCOMs (immunostimulating complexes), which ISCOMS maybe devoid of additional detergent e.g. WO 00/07621; (3) CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4)cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon),macrophage colony stimulating factor (M-CSF), tumor necrosis factor(TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL(3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions (see, e.g. EP-A-0835318, EP-A-0735898,EP-A-0761231); (7) oligonucleotides comprising a CpG motif containing atleast one CG dinucleotide, where the cytosine is unmethylated (see,e.g., WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495,WO 98/37919 and WO 98/52581); (8) a polyoxyethylene ether or apolyoxyethylene ester (see, e.g. WO 99/52549); (9) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (WO 01/21207)or a polyoxyethylene alkyl ether or ester surfactant in combination withat least one additional non-ionic surfactant such as an octoxynol (WO01/21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g.a CpG oligonucleotide) (WO 00/62800); (11) an immunostimulant and aparticle of metal salt (see, e.g. WO 00/23105); (12) a saponin and anoil-in-water emulsion (see e.g. WO 99/11241); (13) a saponin (e.g.QS21)+3dMPL+IM2 (optionally including a sterol) (see, e.g. WO 98/57659);(14) other substances that act as immunostimulating agents to enhancethe efficacy of the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc. Also suitable for use is Matrix-M™; Matrix-M™ is anadjuvant that comprises 40 nm nanoparticles comprising Quillajasaponins, cholesterol, and phospholipid. Adjuvants suitable foradministration to a human are of particular interest. In some cases, theadjuvant is one that enhances a CD4⁺ T helper response to the immunogen.Also suitable for use is a poly inosine:cytosine (poly I:C) nucleicacid. Poly I:C is a synthetic double-stranded RNA Also suitable for useis a cyclic dinucleotide activator of the STING pathway. Examples ofsuitable cyclic dinucleotide adjuvants include, but are not limitedto: 1) bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP); 2)bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP); andbis-(3′,5′)-cyclic dimeric inosine monosphosphate (c-di-IMP).

In some instances, the adjuvant is MF59, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant isalum, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is poly(D,L-lactide-co-glycolide), with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is MPL, with or without a CpG-containing oligonucleotide. Insome cases, the adjuvant is Matrix-M™, with or without a CpG-containingoligonucleotide. In some cases, the adjuvant is keyhole limpethemocyanin. In some cases, the adjuvant is alum. In some cases, theadjuvant is aluminum phosphate. In some cases, the adjuvant is aluminumhydroxide. In some cases, the adjuvant is alum+MPL. In some cases, theadjuvant is MF59. In some cases, the adjuvant is alum+MF59. In somecases, the adjuvant is AS01. AS01 contains QS-21 Stimulon® adjuvant,MPL, and liposomes. In some cases, the adjuvant is AS03. A dose of S03contains: 10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mgpolysorbate-80. In some cases, the adjuvant is AS04. In some cases, theadjuvant is AS15. AS15 is a combination of QS-21 Stimulon® adjuvant,monophosphoryl lipid A, and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; (SEQ ID NO:123), in a liposomalformulation.

Exemplary Compositions

The following are non-limiting examples of immunogenic compositions ofthe present disclosure.

1) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide comprising an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ IDNO:94); and having a length of from 29 amino acids (aa) to 35 aa (e.g.,29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, or 35 aa); and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvant isMF59, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:94); and having a length of 29amino acids; and c) a pharmaceutically acceptable carrier. In somecases, the immunogenic composition comprises an adjuvant. In someinstances, the adjuvant comprises MF59, alum, AS01, AS03, AS04, AS15,MPL, QS-21, or a CpG-containing oligonucleotide (e.g., CpG7909), or acombination of two of the foregoing. In some instances, the adjuvantcomprises MF59. In some instances, the adjuvant comprises alum. In someinstances, the adjuvant comprises alum+MPL. In some instances, theadjuvant comprises QS-21. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 heterodimers from different HCVgenotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

2) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide comprising an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ IDNO:94); and having a length of from 29 amino acids (aa) to 35 aa (e.g.,29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, or 35 aa); and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvant isMF59, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:94); and having a length of 29amino acids; and c) a pharmaceutically acceptable carrier. In somecases, the immunogenic composition comprises an adjuvant. In someinstances, the adjuvant comprises MF59, alum, AS01, AS03, AS04, AS15,MPL, QS-21, or a CpG-containing oligonucleotide (e.g., CpG7909), or acombination of two of the foregoing. In some instances, the adjuvantcomprises MF59. In some instances, the adjuvant comprises alum. In someinstances, the adjuvant comprises alum+MPL. In some instances, theadjuvant comprises QS-21. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 heterodimers from different HCVgenotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

3) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99); andthat has a length of from 50 amino acids to 60 amino acids (e.g., 50 aa,51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60aa); and c) a pharmaceutically acceptable carrier. In some cases, theimmunogenic composition comprises an adjuvant. In some instances, theadjuvant is MF59, with or without a CpG-containing oligonucleotide. Inother instances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99); andhaving a length of 50 amino acids; and c) a pharmaceutically acceptablecarrier. In some cases, the immunogenic composition comprises anadjuvant. In some instances, the adjuvant comprises MF59, alum, AS01,AS03, AS04, AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g.,CpG7909), or a combination of two of the foregoing. In some instances,the adjuvant comprises MF59. In some instances, the adjuvant comprisesalum. In some instances, the adjuvant comprises alum+MPL. In someinstances, the adjuvant comprises QS-21. In some cases, the immunogeniccomposition comprises a mixture of HCV E1/E2 heterodimers from differentHCV genotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

4) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99); andthat has a length of from 50 amino acids to 60 amino acids (e.g., 50 aa,51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60aa); and c) a pharmaceutically acceptable carrier. In some cases, theimmunogenic composition comprises an adjuvant. In some instances, theadjuvant is MF59, with or without a CpG-containing oligonucleotide. Inother instances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99); andhaving a length of 50 amino acids; and c) a pharmaceutically acceptablecarrier. In some cases, the immunogenic composition comprises anadjuvant. In some instances, the adjuvant comprises MF59, alum, AS01,AS03, AS04, AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g.,CpG7909), or a combination of two of the foregoing. In some instances,the adjuvant comprises MF59. In some instances, the adjuvant comprisesalum. In some instances, the adjuvant comprises alum+MPL. In someinstances, the adjuvant comprises QS-21. In some cases, the immunogeniccomposition comprises a mixture of HCV E1/E2 heterodimers from differentHCV genotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

5) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95); andthat has a length of from 52 amino acids to 60 amino acids (e.g., 52 aa,53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa); and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvant isMF59, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95); andhaving a length of 52 amino acids; and c) a pharmaceutically acceptablecarrier. In some cases, the immunogenic composition comprises anadjuvant. In some instances, the adjuvant comprises MF59, alum, AS01,AS03, AS04, AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g.,CpG7909), or a combination of two of the foregoing. In some instances,the adjuvant comprises MF59. In some instances, the adjuvant comprisesalum. In some instances, the adjuvant comprises alum+MPL. In someinstances, the adjuvant comprises QS-21. In some cases, the immunogeniccomposition comprises a mixture of HCV E1/E2 heterodimers from differentHCV genotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

6) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95); andthat has a length of from 52 amino acids to 60 amino acids (e.g., 52 aa,53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa); and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvant isMF59, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95); andhaving a length of 52 amino acids; and c) a pharmaceutically acceptablecarrier. In some cases, the immunogenic composition comprises anadjuvant. In some instances, the adjuvant comprises MF59, alum, AS01,AS03, AS04, AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g.,CpG7909), or a combination of two of the foregoing. In some instances,the adjuvant comprises MF59. In some instances, the adjuvant comprisesalum. In some instances, the adjuvant comprises alum+MPL. In someinstances, the adjuvant comprises QS-21. In some cases, the immunogeniccomposition comprises a mixture of HCV E1/E2 heterodimers from differentHCV genotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

7) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96); and that has a length of from 70 amino acids to 80 aminoacids (e.g., 70 aa, 71 aa, 72 aa, 73 aa, 74 aa, 75 aa, 76 aa, 77 aa, 78aa, 79 aa, or 80 aa); and c) a pharmaceutically acceptable carrier. Insome cases, the immunogenic composition comprises an adjuvant. In someinstances, the adjuvant is MF59, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is alum, with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is poly(D,L-lactide-co-glycolide), with or without aCpG-containing oligonucleotide. In other instances, the adjuvant is MPL,with or without a CpG-containing oligonucleotide. In some cases, theadjuvant is Matrix-M™, with or without a CpG-containing oligonucleotide.In some cases, the adjuvant is alum. In some cases, the adjuvant isMF59. In some cases, the adjuvant is alum+MF59. In some cases, theadjuvant is AS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, andliposomes. In some cases, the adjuvant is AS03. A dose of S03 contains:10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80.In some cases, the adjuvant is AS04. In some cases, the adjuvant isAS15. AS15 is a combination of QS-21 Stimulon® adjuvant, monophosphoryllipid A, and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96); and having a length of 70 amino acids; and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvantcomprises MF59, alum, AS01, AS03, AS04, AS15, MPL, QS-21, or aCpG-containing oligonucleotide (e.g., CpG7909), or a combination of twoof the foregoing. In some instances, the adjuvant comprises MF59. Insome instances, the adjuvant comprises alum. In some instances, theadjuvant comprises alum+MPL. In some instances, the adjuvant comprisesQS-21. In some cases, the immunogenic composition comprises a mixture ofHCV E1/E2 heterodimers from different HCV genotypes. In some cases, theimmunogenic composition comprises a mixture of HCV E1/E2 of genotype 1and HCV E1/E2 of genotype 3. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 of genotype 1, HCV E1/E2 of genotype 2,and HCV E1/E2 of genotype 3.

8) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96); and that has a length of from 70 amino acids to 80 aminoacids (e.g., 70 aa, 71 aa, 72 aa, 73 aa, 74 aa, 75 aa, 76 aa, 77 aa, 78aa, 79 aa, or 80 aa); and c) a pharmaceutically acceptable carrier. Insome cases, the immunogenic composition comprises an adjuvant. In someinstances, the adjuvant is MF59, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is alum, with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is poly(D,L-lactide-co-glycolide), with or without aCpG-containing oligonucleotide. In other instances, the adjuvant is MPL,with or without a CpG-containing oligonucleotide. In some cases, theadjuvant is Matrix-M™, with or without a CpG-containing oligonucleotide.In some cases, the adjuvant is alum. In some cases, the adjuvant isMF59. In some cases, the adjuvant is alum+MF59. In some cases, theadjuvant is AS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, andliposomes. In some cases, the adjuvant is AS03. A dose of S03 contains:10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80.In some cases, the adjuvant is AS04. In some cases, the adjuvant isAS15. AS15 is a combination of QS-21 Stimulon® adjuvant, monophosphoryllipid A, and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96); and having a length of 70 amino acids; and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvantcomprises MF59, alum, AS01, AS03, AS04, AS15, MPL, QS-21, or aCpG-containing oligonucleotide (e.g., CpG7909), or a combination of twoof the foregoing. In some instances, the adjuvant comprises MF59. Insome instances, the adjuvant comprises alum. In some instances, theadjuvant comprises alum+MPL. In some instances, the adjuvant comprisesQS-21. In some cases, the immunogenic composition comprises a mixture ofHCV E1/E2 heterodimers from different HCV genotypes. In some cases, theimmunogenic composition comprises a mixture of HCV E1/E2 of genotype 1and HCV E1/E2 of genotype 3. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 of genotype 1, HCV E1/E2 of genotype 2,and HCV E1/E2 of genotype 3.

9) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97); and that has alength of from 100 amino acids (aa) to 110 aa (e.g., 100 aa, 101 aa, 102aa, 103 aa, 104 aa, 105 aa, 106 aa, 107 aa, 108 aa, 109 aa, or 110 aa);and c) a pharmaceutically acceptable carrier. In some cases, theimmunogenic composition comprises an adjuvant. In some instances, theadjuvant is MF59, with or without a CpG-containing oligonucleotide. Inother instances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97); and having alength of 100 amino acids; and c) a pharmaceutically acceptable carrier.In some cases, the immunogenic composition comprises an adjuvant. Insome instances, the adjuvant comprises MF59, alum, AS01, AS03, AS04,AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g., CpG7909),or a combination of two of the foregoing. In some instances, theadjuvant comprises MF59. In some instances, the adjuvant comprises alum.In some instances, the adjuvant comprises alum+MPL. In some instances,the adjuvant comprises QS-21. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 heterodimers from different HCVgenotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

10) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97); and that has alength of from 100 amino acids (aa) to 110 aa (e.g., 100 aa, 101 aa, 102aa, 103 aa, 104 aa, 105 aa, 106 aa, 107 aa, 108 aa, 109 aa, or 110 aa);and c) a pharmaceutically acceptable carrier. In some cases, theimmunogenic composition comprises an adjuvant. In some instances, theadjuvant is MF59, with or without a CpG-containing oligonucleotide. Inother instances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97); and having alength of 100 amino acids; and c) a pharmaceutically acceptable carrier.In some cases, the immunogenic composition comprises an adjuvant. Insome instances, the adjuvant comprises MF59, alum, AS01, AS03, AS04,AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g., CpG7909),or a combination of two of the foregoing. In some instances, theadjuvant comprises MF59. In some instances, the adjuvant comprises alum.In some instances, the adjuvant comprises alum+MPL. In some instances,the adjuvant comprises QS-21. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 heterodimers from different HCVgenotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

11) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and can has a length of from 171 amino acids (aa) to 180 aa (e.g., 171aa, 172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa, 179 aa, or180 aa); and c) a pharmaceutically acceptable carrier. In some cases,the immunogenic composition comprises an adjuvant. In some instances,the adjuvant is MF59, with or without a CpG-containing oligonucleotide.In other instances, the adjuvant is alum, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and having a length of 171 amino acids; and c) a pharmaceuticallyacceptable carrier. In some cases, the immunogenic composition comprisesan adjuvant. In some instances, the adjuvant comprises MF59, alum, AS01,AS03, AS04, AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g.,CpG7909), or a combination of two of the foregoing. In some instances,the adjuvant comprises MF59. In some instances, the adjuvant comprisesalum. In some instances, the adjuvant comprises alum+MPL. In someinstances, the adjuvant comprises QS-21. In some cases, the immunogeniccomposition comprises a mixture of HCV E1/E2 heterodimers from differentHCV genotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

12) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and can has a length of from 171 amino acids (aa) to 180 aa (e.g., 171aa, 172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa, 179 aa, or180 aa); and c) a pharmaceutically acceptable carrier. In some cases,the immunogenic composition comprises an adjuvant. In some instances,the adjuvant is MF59, with or without a CpG-containing oligonucleotide.In other instances, the adjuvant is alum, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and having a length of 171 amino acids; and c) a pharmaceuticallyacceptable carrier. In some cases, the immunogenic composition comprisesan adjuvant. In some instances, the adjuvant comprises MF59, alum, AS01,AS03, AS04, AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g.,CpG7909), or a combination of two of the foregoing. In some instances,the adjuvant comprises MF59. In some instances, the adjuvant comprisesalum. In some instances, the adjuvant comprises alum+MPL. In someinstances, the adjuvant comprises QS-21. In some cases, the immunogeniccomposition comprises a mixture of HCV E1/E2 heterodimers from differentHCV genotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

13) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:81); and thathas a length of from 228 amino acids (aa) to 235 aa (e.g., 228 aa, 229aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa, or 235 aa); and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvant isMF59, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:

(SEQ ID NO: 81) LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN;

and having a length of 228 amino acids; and c) a pharmaceuticallyacceptable carrier. In some cases, the immunogenic composition comprisesan adjuvant. In some instances, the adjuvant comprises MF59, alum, AS01,AS03, AS04, AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g.,CpG7909), or a combination of two of the foregoing. In some instances,the adjuvant comprises MF59. In some instances, the adjuvant comprisesalum. In some instances, the adjuvant comprises alum+MPL. In someinstances, the adjuvant comprises QS-21. In some cases, the immunogeniccomposition comprises a mixture of HCV E1/E2 heterodimers from differentHCV genotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

14) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 81) LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN;and that has a length of from 228 amino acids (aa) to 235 aa (e.g., 228aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa, 234 aa, or 235 aa); and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvant isMF59, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is alum, with or without a CpG-containingoligonucleotide. In other instances, the adjuvant ispoly(D,L-lactide-co-glycolide), with or without a CpG-containingoligonucleotide. In other instances, the adjuvant is MPL, with orwithout a CpG-containing oligonucleotide. In some cases, the adjuvant isMatrix-M™, with or without a CpG-containing oligonucleotide. In somecases, the adjuvant is alum. In some cases, the adjuvant is MF59. Insome cases, the adjuvant is alum+MF59. In some cases, the adjuvant isAS01. AS01 contains QS-21 Stimulon® adjuvant, MPL, and liposomes. Insome cases, the adjuvant is AS03. A dose of S03 contains: 10.69 mgsqualene; 11.86 mg DL-a-tocopherol; and 4.86 mg polysorbate-80. In somecases, the adjuvant is AS04. In some cases, the adjuvant is AS15. AS15is a combination of QS-21 Stimulon® adjuvant, monophosphoryl lipid A,and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:

(SEQ ID NO: 81) LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN;

and having a length of 228 amino acids; and c) a pharmaceuticallyacceptable carrier. In some cases, the immunogenic composition comprisesan adjuvant. In some instances, the adjuvant comprises MF59, alum, AS01,AS03, AS04, AS15, MPL, QS-21, or a CpG-containing oligonucleotide (e.g.,CpG7909), or a combination of two of the foregoing. In some instances,the adjuvant comprises MF59. In some instances, the adjuvant comprisesalum. In some instances, the adjuvant comprises alum+MPL. In someinstances, the adjuvant comprises QS-21. In some cases, the immunogeniccomposition comprises a mixture of HCV E1/E2 heterodimers from differentHCV genotypes. In some cases, the immunogenic composition comprises amixture of HCV E1/E2 of genotype 1 and HCV E1/E2 of genotype 3. In somecases, the immunogenic composition comprises a mixture of HCV E1/E2 ofgenotype 1, HCV E1/E2 of genotype 2, and HCV E1/E2 of genotype 3.

15) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100); and that has a length of from 553amino acids (aa) to 560 aa (e.g., aa, 554 aa, 555 aa, 556 aa, 557 aa,558 aa, 559 aa, or 560 aa); and c) a pharmaceutically acceptablecarrier. In some cases, the immunogenic composition comprises anadjuvant. In some instances, the adjuvant is MF59, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant isalum, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is poly(D,L-lactide-co-glycolide), with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is MPL, with or without a CpG-containing oligonucleotide. Insome cases, the adjuvant is Matrix-M™, with or without a CpG-containingoligonucleotide. In some cases, the adjuvant is alum. In some cases, theadjuvant is MF59. In some cases, the adjuvant is alum+MF59. In somecases, the adjuvant is AS01. AS01 contains QS-21 Stimulon® adjuvant,MPL, and liposomes. In some cases, the adjuvant is AS03. A dose of S03contains: 10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mgpolysorbate-80. In some cases, the adjuvant is AS04. In some cases, theadjuvant is AS15. AS15 is a combination of QS-21 Stimulon® adjuvant,monophosphoryl lipid A, and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100); and having a length of 553 aminoacids; and c) a pharmaceutically acceptable carrier. In some cases, theimmunogenic composition comprises an adjuvant. In some instances, theadjuvant comprises MF59, alum, AS01, AS03, AS04, AS15, MPL, QS-21, or aCpG-containing oligonucleotide (e.g., CpG7909), or a combination of twoof the foregoing. In some instances, the adjuvant comprises MF59. Insome instances, the adjuvant comprises alum. In some instances, theadjuvant comprises alum+MPL. In some instances, the adjuvant comprisesQS-21. In some cases, the immunogenic composition comprises a mixture ofHCV E1/E2 heterodimers from different HCV genotypes. In some cases, theimmunogenic composition comprises a mixture of HCV E1/E2 of genotype 1and HCV E1/E2 of genotype 3. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 of genotype 1, HCV E1/E2 of genotype 2,and HCV E1/E2 of genotype 3.

16) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100); and that has a length of from 553amino acids (aa) to 560 aa (e.g., aa, 554 aa, 555 aa, 556 aa, 557 aa,558 aa, 559 aa, or 560 aa); and c) a pharmaceutically acceptablecarrier. In some cases, the immunogenic composition comprises anadjuvant. In some instances, the adjuvant is MF59, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant isalum, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is poly(D,L-lactide-co-glycolide), with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is MPL, with or without a CpG-containing oligonucleotide. Insome cases, the adjuvant is Matrix-M™, with or without a CpG-containingoligonucleotide. In some cases, the adjuvant is alum. In some cases, theadjuvant is MF59. In some cases, the adjuvant is alum+MF59. In somecases, the adjuvant is AS01. AS01 contains QS-21 Stimulon® adjuvant,MPL, and liposomes. In some cases, the adjuvant is AS03. A dose of S03contains: 10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mgpolysorbate-80. In some cases, the adjuvant is AS04. In some cases, theadjuvant is AS15. AS15 is a combination of QS-21 Stimulon® adjuvant,monophosphoryl lipid A, and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100); and having a length of 553 aminoacids; and c) a pharmaceutically acceptable carrier. In some cases, theimmunogenic composition comprises an adjuvant. In some instances, theadjuvant comprises MF59, alum, AS01, AS03, AS04, AS15, MPL, QS-21, or aCpG-containing oligonucleotide (e.g., CpG7909), or a combination of twoof the foregoing. In some instances, the adjuvant comprises MF59. Insome instances, the adjuvant comprises alum. In some instances, theadjuvant comprises alum+MPL. In some instances, the adjuvant comprisesQS-21. In some cases, the immunogenic composition comprises a mixture ofHCV E1/E2 heterodimers from different HCV genotypes. In some cases, theimmunogenic composition comprises a mixture of HCV E1/E2 of genotype 1and HCV E1/E2 of genotype 3. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 of genotype 1, HCV E1/E2 of genotype 2,and HCV E1/E2 of genotype 3.

17) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Prodipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Pro-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and that has a length of from 778 amino acids (aa) to790 aa (e.g., 778 aa, 779 aa, 780 aa, 781 aa, 782 aa, 783 aa, 784 aa,785 aa, 786 aa, 787 aa, 788 aa, or 790 aa); and c) a pharmaceuticallyacceptable carrier. In some cases, the immunogenic composition comprisesan adjuvant. In some instances, the adjuvant is MF59, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant isalum, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is poly(D,L-lactide-co-glycolide), with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is MPL, with or without a CpG-containing oligonucleotide. Insome cases, the adjuvant is Matrix-M™, with or without a CpG-containingoligonucleotide. In some cases, the adjuvant is alum. In some cases, theadjuvant is MF59. In some cases, the adjuvant is alum+MF59. In somecases, the adjuvant is AS01. AS01 contains QS-21 Stimulon® adjuvant,MPL, and liposomes. In some cases, the adjuvant is AS03. A dose of S03contains: 10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mgpolysorbate-80. In some cases, the adjuvant is AS04. In some cases, theadjuvant is AS15. AS15 is a combination of QS-21 Stimulon® adjuvant,monophosphoryl lipid A, and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Pro dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Pro-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and having a length of 778 amino acids; and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvantcomprises MF59, alum, AS01, AS03, AS04, AS15, MPL, QS-21, or aCpG-containing oligonucleotide (e.g., CpG7909), or a combination of twoof the foregoing. In some instances, the adjuvant comprises MF59. Insome instances, the adjuvant comprises alum. In some instances, theadjuvant comprises alum+MPL. In some instances, the adjuvant comprisesQS-21. In some cases, the immunogenic composition comprises a mixture ofHCV E1/E2 heterodimers from different HCV genotypes. In some cases, theimmunogenic composition comprises a mixture of HCV E1/E2 of genotype 1and HCV E1/E2 of genotype 3. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 of genotype 1, HCV E1/E2 of genotype 2,and HCV E1/E2 of genotype 3.

18) In some cases, an immunogenic composition of the present disclosurecomprises: a) an HCV E1/E2 heterodimer comprising: i) an HCV E1polypeptide; and ii) a modified HCV E2 polypeptide comprising a Gly-Serdipeptide appended to the N-terminus of an HCV E2 polypeptide (i.e.,where the modified HCV E2 polypeptide is a Gly-Ser-E2 polypeptide); b) apolypeptide that comprises an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and that has a length of from 778 amino acids (aa) to790 aa (e.g., 778 aa, 779 aa, 780 aa, 781 aa, 782 aa, 783 aa, 784 aa,785 aa, 786 aa, 787 aa, 788 aa, or 790 aa); and c) a pharmaceuticallyacceptable carrier. In some cases, the immunogenic composition comprisesan adjuvant. In some instances, the adjuvant is MF59, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant isalum, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is poly(D,L-lactide-co-glycolide), with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is MPL, with or without a CpG-containing oligonucleotide. Insome cases, the adjuvant is Matrix-M™, with or without a CpG-containingoligonucleotide. In some cases, the adjuvant is alum. In some cases, theadjuvant is MF59. In some cases, the adjuvant is alum+MF59. In somecases, the adjuvant is AS01. AS01 contains QS-21 Stimulon® adjuvant,MPL, and liposomes. In some cases, the adjuvant is AS03. A dose of S03contains: 10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mgpolysorbate-80. In some cases, the adjuvant is AS04. In some cases, theadjuvant is AS15. AS15 is a combination of QS-21 Stimulon® adjuvant,monophosphoryl lipid A, and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; SEQ ID NO:123), in a liposomalformulation. In some cases, an immunogenic composition of the presentdisclosure comprises: a) an HCV E1/E2 heterodimer comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aGly-Ser dipeptide appended to the N-terminus of an HCV E2 polypeptide(i.e., where the modified HCV E2 polypeptide is a Gly-Ser-E2polypeptide); b) a polypeptide comprising an amino acid sequence havingat least 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to the following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and having a length of 778 amino acids; and c) apharmaceutically acceptable carrier. In some cases, the immunogeniccomposition comprises an adjuvant. In some instances, the adjuvantcomprises MF59, alum, AS01, AS03, AS04, AS15, MPL, QS-21, or aCpG-containing oligonucleotide (e.g., CpG7909), or a combination of twoof the foregoing. In some instances, the adjuvant comprises MF59. Insome instances, the adjuvant comprises alum. In some instances, theadjuvant comprises alum+MPL. In some instances, the adjuvant comprisesQS-21. In some cases, the immunogenic composition comprises a mixture ofHCV E1/E2 heterodimers from different HCV genotypes. In some cases, theimmunogenic composition comprises a mixture of HCV E1/E2 of genotype 1and HCV E1/E2 of genotype 3. In some cases, the immunogenic compositioncomprises a mixture of HCV E1/E2 of genotype 1, HCV E1/E2 of genotype 2,and HCV E1/E2 of genotype 3.

Compositions Comprising a Polypeptide Comprising T-Cell Epitopes Presentin an HCV Polypeptide Other than E1 and E2

The present disclosure provides an immunogenic composition comprising:a) a polypeptide comprising one or more T-cell epitopes present in anHCV polypeptide other than E1 and E2; and b) a pharmaceuticallyacceptable excipient. For simplicity, a “polypeptide comprising one ormore T-cell epitopes present in an HCV polypeptide other than E1 and E2”is referred to as an “HCV T-cell epitope polypeptide.” In some cases,the immunogenic composition comprises an adjuvant. Thus, the presentdisclosure provides an immunogenic composition comprising: a) an HCVT-cell epitope polypeptide; b) a pharmaceutically acceptable excipient;and c) an adjuvant. An immunogenic composition of the present disclosurethat comprises a polypeptide comprising one or more T-cell epitopespresent in an HCV polypeptide other than E1 and E2 does not include anHCV E1/E2 heterodimer, an HCV E2 polypeptide, or an HCV E1 polypeptide.

In some cases, an HCV T-cell epitope polypeptide is a modified T-cellepitope polypeptide that comprises from 1 to 6 additional amino acids atthe N-terminus of the modified HCV T-cell epitope polypeptide, where thefrom 1 to 6 additional amino acids are Gly-Pro, Ser, Gly, or Gly-Ser. Insome cases, an HCV T-cell epitope polypeptide is a modified HCV T-cellepitope polypeptide that comprises from 1 to 6 additional amino acids atthe C-terminus of the modified HCV T-cell epitope polypeptide, where thefrom 1 to 6 additional amino acids are LEVLFQ (SEQ ID NO:76), ENLYYFQ(SEQ ID NO:83), LVPR (SEQ ID NO:78), I(E/D)GR (SEQ ID NO:90), or DDDDK(SEQ ID NO:77).

In some cases, the HCV T-cell epitope polypeptide does not include aneotope; for example, in some cases, the HCV T-cell epitope polypeptidedoes not include a junction formed by amino acid sequences that do notnaturally occur adjacent to one another in a naturally-occurring HCVpolypeptide.

In some cases, the HCV T-cell epitope polypeptide is a fusionpolypeptide comprising: i) the HCV T-cell epitope polypeptide; and ii) afusion partner polypeptide, where the fusion partner polypeptide cancomprise a T-cell epitope present in a polypeptide other than an HCVpolypeptide. In some cases, the immunogenic composition comprises: a) afusion polypeptide comprising: i) an HCV T-cell epitope polypeptide; andii) a fusion partner polypeptide comprising a T-cell epitope present ina polypeptide other than an HCV polypeptide; and b) a pharmaceuticallyacceptable excipient. In some cases, the immunogenic compositioncomprises: a) a fusion polypeptide comprising: i) an HCV T-cell epitopepolypeptide; and ii) a fusion partner polypeptide comprising a T-cellepitope present in a polypeptide other than an HCV polypeptide; b) apharmaceutically acceptable excipient; and c) an adjuvant.

In some cases, the immunogenic composition comprises: a) an HCV T-cellepitope polypeptide; b) a pharmaceutically acceptable excipient; and c)a non-HCV polypeptide. In some cases, the immunogenic compositioncomprises: a) an HCV T-cell epitope polypeptide; b) a pharmaceuticallyacceptable excipient; c) a non-HCV polypeptide; and d) an adjuvant. Insome cases, the non-HCV polypeptide comprises one or more T-cellepitopes.

As noted above, an immunogenic composition of the present disclosure anHCV T-cell epitope polypeptide comprising a T-cell epitope present in anHCV protein other than E1 and E2.

In some cases, the HCV T-cell epitope polypeptide comprises 1, 2, 3, 4,5, 6, 7, 8, 9, or 10, or more than 10 (e.g., from 10 to 15, from 15 to20, from 20 to 25, or from 25 to 30, or more than 30), T cell epitopes.T-cell epitopes include epitopes recognized by cytotoxic T cells (e.g.,CD8⁺ T cells), and epitopes recognized by helper T cells (e.g., CD4⁺ Tcells).

An immunogenic composition of the present disclosure that comprises anHCV T-cell epitope polypeptide can, when administered to an individualin need thereof, induce a CD4⁺ T cell response and/or a CD8⁺ T cellresponse.

The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the HCV T-cell epitope polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein. A suitable source of T-cellepitopes includes CRM. Other examples of strong T helper epitopes arediphtheria toxoid, tetanus toxoid, meningococcal outer membrane protein,or mutant diphtheria protein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, the HCV T-cell epitope polypeptide comprises a singleT-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-NS3 T-cell epitope. In some cases, the HCV T-cellepitope polypeptide comprises 2 or more T-cell epitopes. In some cases,the HCV T-cell epitope polypeptide comprises 2 or more HCV-NS3 T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 3or more HCV-NS3 T-cell epitopes. In some cases, the HCV T-cell epitopepolypeptide comprises 4 or more HCV-NS3 T-cell epitopes. In some cases,the HCV T-cell epitope polypeptide comprises a single HCV-NS3 CD4⁺T-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises 2 or more HCV-NS3 CD4⁺ T-cell epitopes. In some cases, the HCVT-cell epitope polypeptide comprises one or more HCV CD8⁺ T cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises asingle HCV-NS3 CD8⁺ T-cell epitope. In some cases, the HCV T-cellepitope polypeptide comprises 2 or more HCV-NS3 CD8⁺ T-cell epitopes. Insome cases, the HCV T-cell epitope polypeptide comprises at least oneHCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope. Insome cases, the HCV T-cell epitope polypeptide comprises at least oneHCV-NS3 CD4⁺ T cell epitope and at least one HCV-NS3 CD8⁺ T cellepitope. In some cases, HCV T-cell epitope polypeptide comprises 2 ormore HCV-NS3 CD4⁺ T-cell epitopes and 2 or more HCV-NS3 CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 2,3, 4, or 5 HCV-NS3 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS3 CD8⁺T-cell epitopes.

In some cases, the HCV T-cell epitope polypeptide comprises a singleT-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-NS2 T-cell epitope. In some cases, the HCV T-cellepitope polypeptide comprises 2 or more T-cell epitopes. In some cases,the HCV T-cell epitope polypeptide comprises 2 or more HCV-NS2 T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 3or more HCV-NS2 T-cell epitopes. In some cases, the HCV T-cell epitopepolypeptide comprises 4 or more HCV-NS2 T-cell epitopes. In some cases,the HCV T-cell epitope polypeptide comprises a single HCV-NS2 CD4⁺T-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises 2 or more HCV-NS2 CD4⁺ T-cell epitopes. In some cases, the HCVT-cell epitope polypeptide comprises one or more HCV CD8⁺ T cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises asingle HCV-NS2 CD8⁺ T-cell epitope. In some cases, the HCV T-cellepitope polypeptide comprises 2 or more HCV-NS2 CD8⁺ T-cell epitopes. Insome cases, the HCV T-cell epitope polypeptide comprises at least oneHCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope. Insome cases, the HCV T-cell epitope polypeptide comprises at least oneHCV-NS2 CD4⁺ T cell epitope and at least one HCV-NS2 CD8⁺ T cellepitope. In some cases, HCV T-cell epitope polypeptide comprises 2 ormore HCV-NS2 CD4⁺ T-cell epitopes and 2 or more HCV-NS2 CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 2,3, 4, or 5 HCV-NS2 CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS2 CD8⁺T-cell epitopes.

In some cases, the HCV T-cell epitope polypeptide comprises a singleT-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-NS4A T-cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises 2 or more T-cell epitopes. In somecases, the HCV T-cell epitope polypeptide comprises 2 or more HCV-NS4AT-cell epitopes. In some cases, the HCV T-cell epitope polypeptidecomprises 3 or more HCV-NS4A T-cell epitopes. In some cases, the HCVT-cell epitope polypeptide comprises 4 or more HCV-NS4A T-cell epitopes.In some cases, the HCV T-cell epitope polypeptide comprises a singleHCV-NS4A CD4⁺ T-cell epitope. In some cases, the HCV T-cell epitopepolypeptide comprises 2 or more HCV-NS4A CD4⁺ T-cell epitopes. In somecases, the HCV T-cell epitope polypeptide comprises one or more HCV CD8⁺T cell epitopes. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-NS4A CD8⁺ T-cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises 2 or more HCV-NS4A CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises atleast one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the HCV T-cell epitope polypeptide comprises atleast one HCV-NS4A CD4⁺ T cell epitope and at least one HCV-NS4A CD8⁺ Tcell epitope. In some cases, HCV T-cell epitope polypeptide comprises 2or more HCV-NS4A CD4⁺ T-cell epitopes and 2 or more HCV-NS4A CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 2,3, 4, or 5 HCV-NS4A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS4A CD8⁺T-cell epitopes.

In some cases, the HCV T-cell epitope polypeptide comprises a singleT-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-NS5A T-cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises 2 or more T-cell epitopes. In somecases, the HCV T-cell epitope polypeptide comprises 2 or more HCV-NS5AT-cell epitopes. In some cases, the HCV T-cell epitope polypeptidecomprises 3 or more HCV-NS5A T-cell epitopes. In some cases, the HCVT-cell epitope polypeptide comprises 4 or more HCV-NS5A T-cell epitopes.In some cases, the HCV T-cell epitope polypeptide comprises a singleHCV-NS5A CD4⁺ T-cell epitope. In some cases, the HCV T-cell epitopepolypeptide comprises 2 or more HCV-NS5A CD4⁺ T-cell epitopes. In somecases, the HCV T-cell epitope polypeptide comprises one or more HCV CD8⁺T cell epitopes. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-NS5A CD8⁺ T-cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises 2 or more HCV-NS5A CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises atleast one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the HCV T-cell epitope polypeptide comprises atleast one HCV-NS5A CD4⁺ T cell epitope and at least one HCV-NS5A CD8⁺ Tcell epitope. In some cases, HCV T-cell epitope polypeptide comprises 2or more HCV-NS5A CD4⁺ T-cell epitopes and 2 or more HCV-NS5A CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 2,3, 4, or 5 HCV-NS5A CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5A CD8⁺T-cell epitopes.

In some cases, the HCV T-cell epitope polypeptide comprises a singleT-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-NS5B T-cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises 2 or more T-cell epitopes. In somecases, the HCV T-cell epitope polypeptide comprises 2 or more HCV-NS5BT-cell epitopes. In some cases, the HCV T-cell epitope polypeptidecomprises 3 or more HCV-NS5B T-cell epitopes. In some cases, the HCVT-cell epitope polypeptide comprises 4 or more HCV-NS5B T-cell epitopes.In some cases, the HCV T-cell epitope polypeptide comprises a singleHCV-NS5B CD4⁺ T-cell epitope. In some cases, the HCV T-cell epitopepolypeptide comprises 2 or more HCV-NS5B CD4⁺ T-cell epitopes. In somecases, the HCV T-cell epitope polypeptide comprises one or more HCV CD8⁺T cell epitopes. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-NS5B CD8⁺ T-cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises 2 or more HCV-NS5B CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises atleast one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the HCV T-cell epitope polypeptide comprises atleast one HCV-NS5B CD4⁺ T cell epitope and at least one HCV-NS5B CD8⁺ Tcell epitope. In some cases, HCV T-cell epitope polypeptide comprises 2or more HCV-NS5B CD4⁺ T-cell epitopes and 2 or more HCV-NS5B CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 2,3, 4, or 5 HCV-NS5B CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-NS5B CD8⁺T-cell epitopes.

In some cases, the HCV T-cell epitope polypeptide comprises a singleT-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-core T-cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises 2 or more T-cell epitopes. In somecases, the HCV T-cell epitope polypeptide comprises 2 or more HCV-coreT-cell epitopes. In some cases, the HCV T-cell epitope polypeptidecomprises 3 or more HCV-core T-cell epitopes. In some cases, the HCVT-cell epitope polypeptide comprises 4 or more HCV-core T-cell epitopes.In some cases, the HCV T-cell epitope polypeptide comprises a singleHCV-core CD4⁺ T-cell epitope. In some cases, the HCV T-cell epitopepolypeptide comprises 2 or more HCV-core CD4⁺ T-cell epitopes. In somecases, the HCV T-cell epitope polypeptide comprises one or more HCV CD8⁺T cell epitopes. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-core CD8⁺ T-cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises 2 or more HCV-core CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises atleast one HCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cellepitope. In some cases, the HCV T-cell epitope polypeptide comprises atleast one HCV-core CD4⁺ T cell epitope and at least one HCV-core CD8⁺ Tcell epitope. In some cases, HCV T-cell epitope polypeptide comprises 2or more HCV-core CD4⁺ T-cell epitopes and 2 or more HCV-core CD8⁺ T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 2,3, 4, or 5 HCV-core CD4⁺ T-cell epitopes and 2, 3, 4, or 5 HCV-core CD8⁺T-cell epitopes.

In some cases, the HCV T-cell epitope polypeptide comprises a singleT-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises a single HCV-p7 T-cell epitope. In some cases, the HCV T-cellepitope polypeptide comprises 2 or more T-cell epitopes. In some cases,the HCV T-cell epitope polypeptide comprises 2 or more HCV-p7 T-cellepitopes. In some cases, the HCV T-cell epitope polypeptide comprises 3or more HCV-p7 T-cell epitopes. In some cases, the HCV T-cell epitopepolypeptide comprises 4 or more HCV-p7 T-cell epitopes. In some cases,the HCV T-cell epitope polypeptide comprises a single HCV-p7 CD4⁺ T-cellepitope. In some cases, the HCV T-cell epitope polypeptide comprises 2or more HCV-core CD4⁺ T-cell epitopes. In some cases, the HCV T-cellepitope polypeptide comprises one or more HCV CD8⁺ T cell epitopes. Insome cases, the HCV T-cell epitope polypeptide comprises a single HCV-p7CD8⁺ T-cell epitope. In some cases, the HCV T-cell epitope polypeptidecomprises 2 or more HCV-p7 CD8⁺ T-cell epitopes. In some cases, the HCVT-cell epitope polypeptide comprises at least one HCV CD4⁺ T cellepitope and at least one HCV CD8⁺ T cell epitope. In some cases, the HCVT-cell epitope polypeptide comprises at least one HCV-p7 CD4⁺ T cellepitope and at least one HCV-p7 CD8⁺ T cell epitope. In some cases, HCVT-cell epitope polypeptide comprises 2 or more HCV-p7 CD4⁺ T-cellepitopes and 2 or more HCV-p7 CD8⁺ T-cell epitopes. In some cases, theHCV T-cell epitope polypeptide comprises 2, 3, 4, or 5 HCV-p7 CD4⁺T-cell epitopes and 2, 3, 4, or 5 HCV-p7 CD8⁺ T-cell epitopes.

In some cases, the HCV T-cell epitope polypeptide comprises 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, or 63, of the T-cell epitopes set out in FIG. 9A-9B. In somecases, the HCV T-cell epitope polypeptide comprises from 1 to 3, from 3to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, or from25 to 30 of the T-cell epitopes set out in FIG. 9A-9B. For example, insome cases, the HCV T-cell epitope polypeptide comprises the T-cellepitopes designated NS3-3, NS3-4, and NS3-11 in FIG. 9A-9B and FIG.11A-11N. As another example, in some cases, the HCV T-cell epitopepolypeptide comprises the T-cell epitopes designated NS2-1, NS2-2,NS2-3, NS2-7, and NS2-8 in FIG. 9A-9B and FIG. 11A-11N. As anotherexample, in some cases, the HCV T-cell epitope polypeptide comprises theT-cell epitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 9A-9Band FIG. 11A-11N. As another example, in some cases, the HCV T-cellepitope polypeptide comprises the T-cell epitopes designated NS3-3,NS3-4, NS3-5, NS3-6, NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 9A-9B andFIG. 11A-11N. As another example, in some cases, the HCV T-cell epitopepolypeptide comprises the T-cell epitopes designated NS3-3, NS3-4,NS3-5, NS3-6, NS3-7, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 9A-9Band FIG. 11A-11N. As another example, in some cases, the HCV T-cellepitope polypeptide comprises the T-cell epitopes designated Core-1,Core-2, Core-3, Core-4, Core-5, Core-6, Core-7, Core-8, Core-9, Core-10,Core-11, Core-12, Core-13, Core-14, Core-16, Core-17, Core-18, Core-19,Core-20, Core-21, and Core-22 in FIG. 9A-9B and FIG. 11A-11N. As anotherexample, in some cases, the HCV T-cell epitope polypeptide comprises theT-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 9A-9B and FIG.11A-11N. As another example, in some cases, the HCV T-cell epitopepolypeptide comprises the T-cell epitopes designated NS2-1, NS2-2,NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8, NS3-1, NS3-2, NS3-3, NS3-4,NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG.9A-9B and FIG. 11A-11N. As another example, in some cases, the HCVT-cell epitope polypeptide comprises the T-cell epitopes designatedNS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10,NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4,NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, and NS4b-10 in FIG. 9A-9B andFIG. 11A-11N. As another example, in some cases, the HCV T-cell epitopepolypeptide comprises the T-cell epitopes designated NS3-1, NS3-2,NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12,NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6,NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1, NS5a-2, NS5b-1, and NS5b-2 inFIG. 9A-9B and FIG. 11A-11N. In some cases, the T-cell epitopes arecontiguous. In some cases, any two T-cell epitopes are separated bylinkers (e.g., a linker having a length of from 1 amino acid to about 50amino acids, e.g., from 1 amino acid to 5 amino acids (aa), from 5 aa to10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 40 aa, or from 40 aa to 50 aa).

In some cases, the HCV T-cell epitope polypeptide comprises at least oneHCV CD4⁺ T cell epitope and at least one HCV CD8⁺ T cell epitope, whereepitopes are conserved among HCV genotypes 1 and 2. In some cases, theHCV T-cell epitope polypeptide comprises at least one HCV CD4⁺ T cellepitope and at least one HCV CD8⁺ T cell epitope, where epitopes areconserved among HCV genotypes 1 and 3. In some cases, the HCV T-cellepitope polypeptide comprises at least one HCV CD4⁺ T cell epitope andat least one HCV CD8⁺ T cell epitope, where epitopes are conserved amongHCV genotypes 1, 2, and 3. In some cases, the HCV T-cell epitopepolypeptide comprises at least one HCV CD4⁺ T cell epitope and at leastone HCV CD8⁺ T cell epitope, where epitopes are conserved among HCVgenotypes 1, 2, 3, and 7. In some cases, the HCV T-cell epitopepolypeptide comprises at least one HCV CD4⁺ T cell epitope and at leastone HCV CD8⁺ T cell epitope, where epitopes are conserved among HCVgenotypes 1-7.

The HCV T-cell epitope polypeptide can have a length of from about 10amino acids to about 2000 amino acids; e.g., the HCV T-cell epitopepolypeptide can have a length of from 10 amino acids (aa) to 15 aa, from15 aa to 20 aa, from 20 aa to 25 aa, from 25 aa to 50 aa, from 50 aa to75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200aa, from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa,from 350 aa to 400 aa, from 450 aa to 500 aa, from 500 aa to 550 aa,from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to 700 aa,from 700 aa to 750 aa, or from 750 aa to 800 aa. The HCV T-cell epitopepolypeptide can have a length of from about 25 amino acids to about 2000amino acids, e.g., from about 25 amino acids (aa) to 50 aa, from 50 aato 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350aa, from 350 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa,from 600 aa to 700 aa, from 700 aa to 800 aa, from 800 aa to 900 aa,from 900 aa to 1000 aa, from 1000 aa to 1100 aa, from 1100 aa to 1200aa, from 1200 aa to 1300 aa, from 1300 aa to 1400 aa, from 1400 aa to1500 aa, from 1500 aa to 1600 aa, from 1600 aa to 1700 aa, from 1700 aato 1800 aa, from 1800 aa to 1900 aa, or from 1900 aa to 2000 aa. The HCVT-cell epitope polypeptide can have a length of from about 25 aminoacids to about 3000 amino acids, e.g., from about 25 amino acids (aa) to50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa,from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300 aa,from 300 aa to 350 aa, from 350 aa to 400 aa, from 400 aa to 500 aa,from 500 aa to 600 aa, from 600 aa to 700 aa, from 700 aa to 800 aa,from 800 aa to 900 aa, from 900 aa to 1000 aa, from 1000 aa to 1100 aa,from 1100 aa to 1200 aa, from 1200 aa to 1300 aa, from 1300 aa to 1400aa, from 1400 aa to 1500 aa, from 1500 aa to 1600 aa, from 1600 aa to1700 aa, from 1700 aa to 1800 aa, from 1800 aa to 1900 aa, from 1900 aato 2000 aa, from 2000 aa to 2250 aa, from 2250 aa to 2500 aa, from 2500aa to 2750 aa, or from 2750 aa to 3000 aa.

The HCV T-cell epitope polypeptide can have a length of from about 25amino acids to about 800 amino acids, e.g., from about 25 amino acids(aa) to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300aa, from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The HCV T-cell epitope polypeptide can have a length of from about 25amino acids to about 400 amino acids, e.g., from about 25 amino acids(aa) to 50 aa, from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to 300aa, from 300 aa to 350 aa, or from 350 aa to 400 aa. The HCV T-cellepitope polypeptide can have a length of 25 amino acids (aa), 26 aa, 27aa, 28 aa, 29 aa, 30 aa, 31 aa, 32 aa, 33 aa, 34 aa, 35 aa, 36 aa, 37aa, 38 aa, 39 aa, 40 aa, 41 aa, 42 aa, 43 aa, 44 aa, 45 aa, 46 aa, 47aa, 48 aa, 49 aa, or 50 aa. The HCV T-cell epitope polypeptide can havea length of from about 100 amino acids (aa) to 800 aa, e.g., from 100 aato 150 aa, from 150 aa to 200 aa, from 200 aa to 250 aa, from 250 aa to300 aa, from 300 aa to 350 aa, from 350 aa to 400 aa, from 450 aa to 500aa, from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 800 aa.The HCV T-cell epitope polypeptide can have a length of from 25 aa to 30aa. The HCV T-cell epitope polypeptide can have a length of from 30 aato 40 aa. The HCV T-cell epitope polypeptide can have a length of from40 aa to 50 aa. The HCV T-cell epitope polypeptide can have a length offrom 50 aa to 60 aa (e.g., 50 aa, 51 aa, 52, aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). The HCV T-cell epitope polypeptidecan have a length of from 60 aa to 70 aa. The HCV T-cell epitopepolypeptide can have a length of from 65 aa to 75 aa (e.g., 65, 66, 67,68, 69, 70, 71, 72, 7, 74, or 75 aa). The HCV T-cell epitope polypeptidecan have a length of 70 aa. The HCV T-cell epitope polypeptide can havea length of from 70 aa to 80 aa. The HCV T-cell epitope polypeptide canhave a length of from 80 aa to 90 aa. The HCV T-cell epitope polypeptidecan have a length of from 90 aa to 100 aa. The HCV T-cell epitopepolypeptide can have a length of from 100 aa to 105 aa (e.g., 100, 101,102, 103, 104, 105, 106, 107, 108, 109, or 110 aa). The HCV T-cellepitope polypeptide can have a length of 100 aa. The HCV T-cell epitopepolypeptide can have a length of from 10 amino acids (aa) to 50 aa;e.g., from 10 aa to 15 aa, from 15 aa to 20 aa, from 20 aa to 25 aa,from 25 aa to 30 aa, from 30 aa to 35 aa, from 35 aa to 40 aa, from 40aa to 45 aa, or from 45 aa to 50 aa. The HCV T-cell epitope polypeptidecan have a length of from 10 amino acids (aa) to 20 aa, e.g., 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 aa.

HCV NS3 T-Cell Epitopes

In some cases, the HCV T-cell epitope polypeptide includes one or moreT-cell epitopes present in an HCV NS3 polypeptide. Examples of T-cellepitopes present in NS3 polypeptides are depicted in FIG. 11A-11N, FIG.9B, and FIG. 10A-10B.

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:94).AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:94) is referred to in FIG. 10Aas “TP29.” In some cases, the HCV T-cell epitope polypeptide comprisesan amino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ ID NO:94); and has a length of from25 aa to 35 aa (e.g., 25 aa, 26 aa, 27 aa, 28 aa, 29 aa, 30 aa, 31 aa,32 aa, 33 aa, 34 aa, or 35 aa). In some cases, the HCV T-cell epitopepolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence: AIPLEVIKGGRHLIFCHSKKKCDELAAKL (SEQ IDNO:94); and has a length of 29 amino acids. Such a polypeptide caninclude NS3 T-cell epitopes designated NS3-3, NS3-4, and NS3-11 in FIG.9B and FIG. 11A-11N.

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95).AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95) isreferred to in FIG. 10A as “TP52.” In some cases, the HCV T-cell epitopepolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95); andhas a length of from 45 amino acids to 60 amino acids (e.g., 45 aa, 46aa, 47 aa, 48 aa, 49 aa, 50 aa, 51 aa, 52 aa, 53 aa, 54 aa, 55 aa, 56aa, 57 aa, 58 aa, 59 aa, or 60 aa). In some cases, the HCV T-cellepitope polypeptide comprises an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity tothe following amino acid sequence:AIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSG (SEQ ID NO:95); andhas a length of 52 amino acids. Such a polypeptide can include NS3T-cell epitopes designated NS3-3, NS3-4, NS3-5, and NS3-11 in FIG. 9Band FIG. 11A-11N

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96); and has a length of from 65 amino acids to 80 aminoacids (e.g., 65 aa, 66 aa, 67 aa, 68 aa, 69 aa, 70 aa, 71 aa, 72 aa, 73aa, 74 aa, 75 aa, 76 aa, 77 aa, 78 aa, 79 aa, or 80 aa).KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96) is referred to in FIG. 10A as “TP70.”

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:KGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTG FTGDFDSVIDCN(SEQ ID NO:96); and has a length of 70 amino acids. Such a polypeptidecan include NS3 T-cell epitopes designated NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-11, NS3-12, and NS3-13 in FIG. 9B and FIG. 11A-11N.

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97); and has alength of from 95 amino acids (aa) to 105 aa (e.g., 95 aa, 96 aa, 97 aa,98 aa, 99 aa, 100 aa, 101 aa, 102 aa, 103 aa, 104 aa, or 105 aa).VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97) is referred toin FIG. 10A as “TP100.”

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:VALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF (SEQ ID NO:97); and has alength of 100 amino acids. Such a polypeptide can include NS3 T-cellepitopes designated NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-10, NS3-11,NS3-12, and NS3-13 in FIG. 9B and FIG. 11A-11N.

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and has a length of from 171 amino acids (aa) to 180 aa (e.g., 171 aa,172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa, 179 aa, or 180aa. MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89)is referred to in FIG. 10A as “TP171.”

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:89);and has a length of 171 amino acids.

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:98); and has a length of from 190 aminoacids (aa) to 200 aa (e.g., 190 aa, 191 aa, 192 aa, 193 aa, 194 aa, 195aa, 196 aa, 197 aa, 198 aa, 199 aa, or 200 aa.

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTVPASA (SEQ ID NO:98); and has a length of 191 amino acids.

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:

(SEQ ID NO: 81) LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN;

and has a length of from 215 amino acids (aa) to 235 aa (e.g., 215 aa,216 aa, 217 11, 218 aa, 219 aa, 220 aa, 221 aa, 222 aa, 223 aa, 224 aa,225 aa, 226 aa, 227 aa, 228 aa, 229 aa, 230 aa, 231 aa, 232 aa, 233 aa,234 aa, or 235 aa).

(SEQ ID NO: 81) LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN;is referred to in FIG. 10A as “TP228.”

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:

(SEQ ID NO: 81) LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN;

and has a length of 228 amino acids. Such a polypeptide can include NS3T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6,NS3-7, NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 in FIG. 9B and FIG.11A-11N.

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1265-1279 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1309-1323 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1401-1415 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1402-1412 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from11 amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa,15 aa, or 16 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1429-1439 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from11 amino acids (aa) to 16 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa,15 aa, or 16 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1450-1464 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1453-1467 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1577-1591 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa,19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1306-1314 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1387-1394 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 1amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1405-1413 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1450-1458 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1457-1465 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV NS3 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1610-1618 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS3 amino acid sequence of anyHCV genotype; and the HCV NS3 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS2 T-Cell Epitopes

In some cases, the HCV T-cell epitope polypeptide includes one or moreT-cell epitopes present in an HCV NS2 polypeptide. Examples of T-cellepitopes present in NS2 polypeptides are depicted in FIG. 11A-11N, andFIG. 9A.

For example, the HCV T-cell epitope polypeptide can comprise an NS2 Tcell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 955-974 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS2 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 975-994 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 20 aminoacids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24 aa,or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS2 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 985-1004 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and the NS2 T cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS2 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1015-1034 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and the NS2 T cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS2 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1035-1054 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and the NS2 T cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS2 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 924-933 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS2 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 961-970 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS2 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 989-997 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of any HCVgenotype; and the NS2 T cell epitope can have a length of from 9 aminoacids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14aa, or 15 aa).

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 10 aminoacids (aa) to 50 aa (e.g., from 10 aa to 25 aa, or from 25 aa to 50 aa)of amino acids 955-1004 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV NS2 amino acidsequence of any HCV genotype; and has a length of from 10 amino acids(aa) to 25 aa, or from 25 aa to 50 aa. In some cases, the HCV T-cellepitope polypeptide comprises an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 955-1004 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS2 amino acid sequence of anyHCV genotype; and has a length of about 50 amino acids.

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 10 aminoacids (aa) to 553 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, or from 500 aa to 553 aa) ofamino acids 917-1469 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS2 and NS3 amino acid sequenceof any HCV genotype; and has a length of from 10 amino acids (aa) to 25aa, from 25 aa to 50 aa, from 50 aa to 100 aa, from 100 aa to 200 aa,from 200 aa to 300 aa, from 300 aa to 400 aa, from 400 aa to 500 aa, orfrom 500 aa to 553 aa. In some cases, the HCV T-cell epitope polypeptidecomprises an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids 917-1469of the amino acid sequence designated “Consensus” in FIG. 12A-12L, or acorresponding HCV NS2 and NS3 amino acid sequence of any HCV genotype;and has a length of about 553 amino acids.

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 0%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99).LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99) isreferred to in FIG. 10A as “TP50.” In some cases, the HCV T-cell epitopepolypeptide comprises an amino acid sequence having at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to thefollowing amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99); andhas a length of from 50 amino acids to 60 amino acids (e.g., 50 aa, 51aa, 52 aa, 53 aa, 54 aa, 55 aa, 56 aa, 57 aa, 58 aa, 59 aa, or 60 aa).In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:LGALTGTYVYNHLTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADT (SEQ ID NO:99); andhas a length of 50 amino acids. Such a polypeptide can include NS2T-cell epitopes designated NS2-1, NS2-2, NS2-3, NS2-7, and NS2-8 in FIG.9A and FIG. 11A-11N.

HCV NS4A T-Cell Epitopes

In some cases, the HCV T-cell epitope polypeptide includes one or moreT-cell epitopes present in an HCV NS4A polypeptide. Examples of T-cellepitopes present in NS4A polypeptides are depicted in FIG. 11A-11N andFIG. 9B.

The HCV T-cell epitope polypeptide can comprise an NS4A T cell epitopecomprising an amino acid sequence having at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to amino acids1683-1692 of the amino acid sequence designated “Consensus” in FIG.12A-12L, or a corresponding HCV NS4A amino acid sequence of any HCVgenotype; and the NS4A T-cell epitope can have a length of from 10 aminoacids (aa) to 15 amino acids (e.g., 10 aa, 11 aa, 12 aa, 13 aa, 14 aa,or 15 aa).

HCV NS4B T-Cell Epitopes

In some cases, the HCV T-cell epitope polypeptide includes one or moreT-cell epitopes present in an HCV NS4B polypeptide. Examples of T-cellepitopes present in NS4B polypeptides are depicted in FIG. 11A-11N andFIG. 9B.

As one example, the HCV T-cell epitope polypeptide can comprise an NS4BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 1790-1801 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of any HCVgenotype; and the NS4B T-cell epitope can have a length of from 12 aminoacids (aa) to 20 amino acids (e.g., 12 aa, 13 aa, 14 aa, 15 aa, 16 aa,17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1792-1802 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 11amino acids (aa) to 20 amino acids (e.g., 11 aa, 12 aa, 13 aa, 14 aa, 15aa, 16 aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1898-1905 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 8amino acids (aa) to 15 amino acids (e.g., 8 aa, 9 aa, 10 aa, 11 aa, 12aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1921-1935 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1922-1941 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1928-1947 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1868-1876 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1927-1942 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 16amino acids (aa) to 20 amino acids (e.g., 16 aa, 17 aa, 18 aa, 19 aa, or20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1932-1940 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS4B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1948-1962 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS4B amino acid sequence of anyHCV genotype; and the NS4B T-cell epitope can have a length of from 15amino acids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19aa, or 20 aa).

HCV NS5A T-Cell Epitopes

In some cases, the HCV T-cell epitope polypeptide includes one or moreT-cell epitopes present in an HCV NS5A polypeptide. Examples of T-cellepitopes present in NS5A polypeptides are depicted in FIG. 11A-11N andFIG. 9B.

As one example, the HCV T-cell epitope polypeptide can comprise an NS5AT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2218-2232 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS5A amino acid sequence of any HCVgenotype; and the NS5A T-cell epitope can have a length of from 15 aminoacids (aa) to 20 amino acids (e.g., 15 aa, 16 aa, 17 aa, 18 aa, 19 aa,or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS5A T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 2309-2317 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS5A amino acid sequence of anyHCV genotype; and the NS5A T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV NS5B T-Cell Epitopes

In some cases, the HCV T-cell epitope polypeptide includes one or moreT-cell epitopes present in an HCV NS5B polypeptide. Examples of T-cellepitopes present in NS5B polypeptides are depicted in FIG. 11A-11N andFIG. 9B.

As one example, the HCV T-cell epitope polypeptide can comprise an NS5BT cell epitope comprising an amino acid sequence having at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about60%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to aminoacids 2847-2851 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV NS5B amino acid sequence of any HCVgenotype; and the NS5B T-cell epitope can have a length of from 5 aminoacids (aa) to 10 amino acids (e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10aa).

As another example, the HCV T-cell epitope polypeptide can comprise anNS5B T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 2602-2610 of the amino acid sequence designated “Consensus”in FIG. 12A-12L, or a corresponding HCV NS5B amino acid sequence of anyHCV genotype; and the NS5B T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV Core T-Cell Epitopes

In some cases, the HCV T-cell epitope polypeptide includes one or moreT-cell epitopes present in an HCV core polypeptide. Examples of T-cellepitopes present in HCV Core polypeptides are depicted in FIG. 11A-11Nand FIG. 9A.

As one example, the HCV T-cell epitope polypeptide can comprise an HCVcore T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 1-20 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV core amino acid sequence of any HCVgenotype; and the HCV core T-cell epitope can have a length of from 20amino acids (aa) to 25 amino acids (e.g., 20 aa, 21 aa, 22 aa, 23 aa, 24aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 11-30 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 21-40 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 39-63 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 23 amino acids (aa) to 28 amino acids (e.g., 23 aa, 24aa, 25 aa, 26 aa, 27 aa, or 28 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 47-70 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 24 amino acids (aa) to 29 amino acids (e.g., 24 aa, 25aa, 26 aa, 27 aa, 28 aa, or 29 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 61-80 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 71-90 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 81-100 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 91-110 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 101-115 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 111-130 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 125-139 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 15 amino acids (aa) to 20 amino acids (e.g., 15 aa, 16aa, 17 aa, 18 aa, 19 aa, or 20 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 131-150 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 151-170 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 161-180 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 20 amino acids (aa) to 25 amino acids (e.g., 20 aa, 21aa, 22 aa, 23 aa, 24 aa, or 25 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 35-44 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 10 amino acids (aa) to 15 amino acids (e.g., 10 aa, 11aa, 12 aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 43-51 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 9 amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa,11 aa, 12 aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 51-59 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 9 amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa,11 aa, 12 aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 129-137 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 9 amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa,11 aa, 12 aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 131-140 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 10 amino acids (aa) to 15 amino acids (e.g., 10 aa, 11aa, 12 aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 150-158 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 9 amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa,11 aa, 12 aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 154-162 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 9 amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa,11 aa, 12 aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 168-176 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 9 amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa,11 aa, 12 aa, 13 aa, 14 aa, or 15 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 177-187 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 11 amino acids (aa) to 16 amino acids (e.g., 11 aa, 12aa, 13 aa, 14 aa, 15 aa, or 16 aa).

As another example, the HCV T-cell epitope polypeptide can comprise anHCV core T cell epitope comprising an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 178-187 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and the HCV core T-cell epitope can have alength of from 10 amino acids (aa) to 15 amino acids (e.g., 10 aa, 11aa, 12 aa, 13 aa, 14 aa, or 15 aa).

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 10 aminoacids (aa) to 191 aa (e.g., from 10 aa to 25 aa, from 25 aa to 50 aa,from 50 aa to 75 aa, from 75 aa to 100 aa, from 100 aa to 150 aa, orfrom 150 aa to 191aa) of amino acids 1-191 of the amino acid sequencedesignated “Consensus” in FIG. 12A-12L, or a corresponding HCV coreamino acid sequence of any HCV genotype; and has a length of from 10amino acids (aa) to 25 aa, from 25 aa to 50 aa, from 50 aa to 100 aa, orfrom 100 aa to 150 aa, or from 150 aa to 191 aa. In some cases, the HCVT-cell epitope polypeptide comprises an amino acid sequence having atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 1-191 of the amino acid sequence designated“Consensus” in FIG. 12A-12L, or a corresponding HCV core amino acidsequence of any HCV genotype; and has a length of about 191 amino acids.

The HCV T-cell epitope polypeptide can comprise an amino acid sequencehaving at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:97);and has a length of from 171 amino acids (aa) to 180 aa (e.g., 171 aa,172 aa, 173 aa, 174 aa, 175 aa, 176 aa, 177 aa, 178 aa, 179 aa, or 180aa. In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPG (SEQ ID NO:97);and has a length of 171 amino acids. Such a polypeptide can include coreT-cell epitopes designated Core-1, Core-2, Core-3, Core-4, Core-5,Core-6, Core-7, Core-8, Core-9, Core-10, Core-11, Core-12, Core-13,Core-14, Core-16, Core-17, Core-18, Core-19, Core-20, Core-21, Core-22in FIG. 9A and FIG. 11A-11N.

HCV p7 T-Cell Epitopes

In some cases, the HCV T-cell epitope polypeptide includes one or moreT-cell epitopes present in an HCV p7 polypeptide. Examples of T-cellepitopes present in HCV p7 polypeptides are depicted in FIG. 11A-11N orFIG. 9A.

As another example, the HCV T-cell epitope polypeptide can comprise anHCV p7 T cell epitope comprising an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toamino acids 803-811 of the amino acid sequence designated “Consensus” inFIG. 12A-12L, or a corresponding HCV p7 amino acid sequence of any HCVgenotype; and the HCV p7 T-cell epitope can have a length of from 9amino acids (aa) to 15 amino acids (e.g., 9 aa, 10 aa, 11 aa, 12 aa, 13aa, 14 aa, or 15 aa).

HCV T-Cell Epitope Polypeptides Including HCV T-Cell Epitopes from Morethan One HCV Polypeptide Other than E1 and E2

As noted above, a HCV T-cell epitope polypeptide can include T-cellepitopes from more than one HCV polypeptide other than E1 and E2.

As one example, a HCV T-cell epitope polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100); and has a length of from 550amino acids (aa) to 560 aa (e.g., 550 aa, 551 aa, 552 aa, 553 aa, 554aa, 555 aa, 556 aa, 557 aa, 558 aa, 559 aa, or 560 aa).

QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100) is referred to in FIG. 10A-10Bas “TP553.”

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:QASLLKVPYFVRVQGLLRICALARKMAGGHYVQMAIIKLGALTGTYVYNALTPLRDWAHNGLRDLAVAVEPVVFSQMETKLITWGADTAACGDIINGLPVSARRGREILLGPADGMVSKGWRLLAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCN (SEQ ID NO:100); and has a length of 553 aminoacids. Such a polypeptide can include T-cell epitopes designated NS2-1,NS2-2, NS2-3, NS2-4, NS2-5, NS2-6, NS2-7, NS2-8, NS3-1, NS3-2, NS3-3,NS3-4, NS3-5, NS3-6, NS3-7, NS3-9, NS3-10, NS3-11, NS3-12, and NS3-13 inFIG. 9A-9B and FIG. 11A-11N. This polypeptide is also referred to as“TP553” (FIG. 12A-12D). In order to prevent self cleavage of the TP553polypeptide (amino acids 917-1469) (FIG. 11E-11G) at the NS2-NS3junction that is mediated by the catalytic domain of the NS2 protease(amino acids 917-1040), the histidine at position 966 (H966), a criticalresidue for NS2 protease activity, is mutated to alanine (H966A) (FIG.11E). See, e.g., Grakoui, A. et al. A second hepatitis C virus-encodedproteinase. Proc. Natl Acad. Sci. USA 90, 10583-10587 (1993); Hijikata,M. et al. Two distinct proteinase activities required for the processingof a putative nonstructural precursor protein of hepatitis C virus. J.Virol. 67, 4665-4675 (1993); and Lorenz. I C. Structure of the catalyticdomain of the hepatitis C virus NS2-3 protease. Nature. August 17;442(7104):831-5 (2006).

As another example, the HCV T-cell epitope polypeptide can comprise anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from 25amino acids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa,from 200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa,from 350 aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa,from 500 aa to 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa,from 650 aa to 700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa)the following amino acid sequence:

(SEQ ID NO: 101) LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPG.

LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPG (SEQ ID NO:101) is referred to in FIG. 10B as “TP778.”

In some cases, the HCV T-cell epitope polypeptide comprises an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from 25 aminoacids (aa) to 778 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75 aa,from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa, from200 aa to 250 aa, from 250 aa to 300 aa, from 300 aa to 350 aa, from 350aa to 400 aa, from 400 aa to 450 aa, from 450 aa to 500 aa, from 500 aato 550 aa, from 550 aa to 600 aa, from 600 aa to 650 aa, from 650 aa to700 aa, from 700 aa to 750 aa, or from 750 aa to 778 aa) of thefollowing amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and has a length of from 25 amino acids (aa) to 50 aa,from 50 aa to 100 aa, from 100 aa to 200 aa, from 200 aa to 300 aa, from300 aa to 400 aa, from 400 aa to 500 aa, from 500 aa to 600 aa, from 600aa to 700 aa, or from 700 aa to 778 aa. In some cases, the HCV T-cellepitope polypeptide comprises an amino acid sequence having at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 60%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity tothe following amino acid sequence:LHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTW LKAKLMPQLPG(SEQ ID NO:101); and has a length of 778 amino acids. Such a polypeptidecan include T-cell epitopes designated NS3-1, NS3-2, NS3-3, NS3-4,NS3-5, NS3-6, NS3-7, NS3-8, NS3-9, NS3-10, NS3-11, NS3-12, NS3-13,NS2-14, NS4a-1, NS4b-1, NS4b-2, NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7,NS4b-8, NS4b-9, and NS4b-10 in FIG. 9B and FIG. 11A-11N.

As another example, the HCV T-cell epitope polypeptide can comprise anamino acid sequence having at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from 25amino acids (aa) to 1985 aa (e.g., from 25 aa to 50 aa, from 50 aa to 75aa, from 75 aa to 100 aa, from 100 aa to 150 aa, from 150 aa to 200 aa,from 200 aa to 250 aa, from 250 aa to 500 aa, from 500 aa to 750 aa,from 750 aa to 1000 aa, from 1000 aa to 1500 aa, or from 1500 aa to 1985aa) of the following amino acid sequence:

(SEQ ID NO: 102) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGS A GGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAA GLQDCTMLVCG NNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR.

In some cases, the HCV T-cell epitope polypeptide can comprise an aminoacid sequence having at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to the following amino acid sequence:APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSFQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLADGGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAYQATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLYREFDEMEECSQHLPYIEQGMMLAEQFKQKALGLLQTASRQAEVIAPAVQTNWQKLEAFWAKHMWNFISGIQYLAGLSTLPGNPAIASLMAFTAAVTSPLTTSQTLLFNILGGWVAAQLAAPGAATAFVGAGLAGAAIGSVGLGKVLVDILAGYGAGVAGALVAFKIMSGEVPSTEDLVNLLPAILSPGALVVGVVCAAILRRHVGPGEGAVQWMNRLIAFASRGNHVSPTHYVPESDAAARVTAILSSLTVTQLLRRLHQWISSECTTPCSGSWLRDIWDWICEVLSDFKTWLKAKLMPQLPGIPFVSCQRGYRGVWRGDGIMHTRCHCGAEITGHVKNGTMRIVGPRTCRNMWSGTFPINAYTTGPCTPLPAPNYTFALWRVSAEEYVEIRQVGDFHYVTGMTTDNLKCPCQVPSPEFFTELDGVRLHRFAPPCKPLLREEVSFRVGLHEYPVGSQLPCEPEPDVAVLTSMLTDPSHITAEAAGRRLARGSPPSVASSSASQLSAPSLKATCTANHDSPDAELIEANLLWRQEMGGNITRVESENKVVILDSFDPLVAEEDEREISVPAEILRKSRRFAPALPIWARPDYNPPLLETWKKPDYEPPVVHGCPLPPPQSPPVPPPRKKRTVVLTESTVSTALAELATKSFGSSSTSGITGDNTTTSSEPAPSGCPPDSDAESYSSMPPLEGEPGDPDLSDGSWSTVSSEADTEDVVCCSMSYSWTGALVTPCAAEEQKLPINALSNSLLRHHNLVYSTTSRSACQRQKKVTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPHSAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEKGGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGPLTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGNNLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSSNVSVAHDGAGKRVYYLTRDPTTPLARAAWETARHTPVNSWLGNIIMFAPTLWARMILMTHFFSVLIARDQLEQALDCEIYGACYSIEPLDLPPIIQRLHGLSAFSLHSYSPGEINRVAACLRKLGVPPLRAWRHRARSVRARLLSRGGRAAICGKYLFNWAVRTKLKLTPIAAAGQLDLSGWFTAGYSGGDIYHSVSHARPRWFWFCLLLLAAGVGIYLLPNR (SEQ ID NO:102); and has a length of1985 amino acids. Such a polypeptide can include T-cell epitopesdesignated NS3-1, NS3-2, NS3-3, NS3-4, NS3-5, NS3-6, NS3-7, NS3-8,NS3-9, NS3-10, NS3-11, NS3-12, NS3-13, NS3-14, NS4a-1, NS4b-1, NS4b-2,NS4b-3, NS4b-4, NS4b-5, NS4b-6, NS4b-7, NS4b-8, NS4b-9, NS4b-10, NS5a-1,NS5a-2, NS5b-1, NS5b-2 in FIG. 9A-9B and FIG. 11A-11N.

Additional T-Cell Epitopes

As discussed above, an immunogenic composition of the present disclosureincludes an HCV T-cell epitope polypeptide that comprises one or moreT-cell epitopes (e.g., one or more T cell epitopes present in an HCVpolypeptide other than an HCV E1 polypeptide or an HCV E2 polypeptide).The one or more T-cell epitopes can include one or more T-cell epitopespresent in: a) an HCV NS3 polypeptide; b) an HCV NS2 polypeptide; c) anHCV NS4A polypeptide; d) an HCV NS4B polypeptide; e) an HCV NS5Apolypeptide; f) an HCV NS5B polypeptide; g) an HCV core polypeptide; orh) an HCV p7 polypeptide. In some cases, the one or more T-cell epitopesare T-cell epitopes present in an HCV NS3 polypeptide. In some cases,the HCV T-cell epitope polypeptide further comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) ameningococcal outer membrane protein.

Thus, in some cases, an immunogenic composition of the presentdisclosure includes: a) an HCV T-cell epitope polypeptide that comprisesone or more T-cell epitopes, where the one or more T-cell epitopes areT-cell epitopes present in: i) one or more of an HCV NS3 polypeptide, anHCV NS2 polypeptide, an HCV NS4A polypeptide, an HCV NS4B polypeptide,an HCV NS5A polypeptide, an HCV NS5B polypeptide, an HCV corepolypeptide, and an HCV p7 polypeptide; and ii) one or more of choleratoxin or toxoid, tetanus toxin or toxoid, diphtheria toxin or toxoid,and a meningococcal outer membrane protein.

A T helper tetanus toxin epitope or other bacterial T-cell epitope couldbe fused (e.g., by recombinant expression) or chemically conjugated tothe HCV T-cell epitope polypeptide, or can be unconjugated (e.g.,provided as a separate polypeptide), to further enhance both T and Bcell responses to both the T-cell epitopes present in the HCV T-cellepitope polypeptide. Alternatively, the whole or part of the detoxifiedtoxin (“toxoid”) can be used, wherein specific amino acids of the toxinsare mutated to render the toxins inactive, thereby generating toxoids.Methods of generating toxoids are well known in the art. Examples ofbacterial epitopes include the use of diphtheria toxoid, meningococcalouter membrane protein, or mutant diphtheria protein CRM197 (see, e.g.:http://www(dot)medscape(dot)com/viewarticle/431127).

In some cases, a suitable tetanus toxoid polypeptide comprises the aminoacid sequence QYIKANSKFIGIFE (SEQ ID NO:103). In some cases, a suitabletetanus toxoid polypeptide comprises the amino acid sequenceQYIKANSKFIGITE (SEQ ID NO:104).

In some cases, a HCV T-cell epitope polypeptide can comprise choleratoxin (or toxoid) epitope. In some cases, a suitable HCV T-cell epitopepolypeptide comprising a cholera toxoid epitope comprises a fragment ofcholera toxin-B subunit (CT-B), e.g., a fragment of from 5 amino acidsto 25 amino acids, or from 25 amino acids to 50 amino acids, of thefollowing amino acid sequence: MIKLKFGVFF TVLLSSAYAH GTPQNITDLCAEYHNTQIHT LNDKIFSYTE SLAGKREMAI ITFKNGATFQ VEVPGSQHID SQKKAIERMKDTLRIAYLTE AKVEKLCVWN NKTPHAIAAI SMAN (SEQ ID NO:105).

In some cases, a HCV T-cell epitope polypeptide can comprise a tetanustoxin (or toxoid) T-cell epitope. In some cases, a suitable HCV T-cellepitope polypeptide comprising a tetanus toxin T-cell epitope comprisesthe amino acid sequence: ILMQYIKANSKFIGI (SEQ ID NO:106); and has alength of from 15 amino acids to 20 amino acids. In some cases, asuitable HCV T-cell epitope polypeptide comprising a tetanus toxinT-cell epitope comprises the amino acid sequence: VNNESSE (SEQ IDNO:107). In some cases, a suitable HCV T-cell epitope polypeptidecomprising a tetanus toxin T-cell epitope comprises the amino acidsequence: PGINGKAIHLVNNESSE (SEQ ID NO:108). In some cases, a suitableHCV T-cell epitope polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: PNRDIL (SEQ ID NO:109). In somecases, a suitable HCV T-cell epitope polypeptide comprising a tetanustoxin T-cell epitope comprises the amino acid sequence: FIGITEL (SEQ IDNO:110). In some cases, a suitable tetanus toxin T-cell epitopecomprises the amino acid sequence: SYFPSV (SEQ ID NO:111). In somecases, a suitable HCV T-cell epitope polypeptide comprising a tetanustoxin T-cell epitope comprises the amino acid sequence:NSVDDALINSTKIYSYFPSV (SEQ ID NO:112). In some cases, a suitable HCVT-cell epitope polypeptide comprising a tetanus toxin T-cell epitopecomprises the amino acid sequence: IDKISDVSTIVPYIGPALNI (SEQ ID NO:113).

In some cases, a HCV T-cell epitope polypeptide can comprise adiphtheria toxin T-cell epitope In some cases, a suitable HCV T-cellepitope polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: QSIALSSLMVAQAIP (SEQ ID NO:114); andhas a length of from 15 amino acids to 20 amino acids. In some cases, asuitable HCV T-cell epitope polypeptide comprising a diphtheria toxinT-cell epitope comprises the amino acid sequence: PVFAGANYAAWAVNVAQVI(SEQ ID NO:115). In some cases, a suitable HCV T-cell epitopepolypeptide comprising a diphtheria toxin T-cell epitope comprises theamino acid sequence: VHHNTEEIVAQSIALSSLMV (SEQ ID NO:116). In somecases, a suitable HCV T-cell epitope polypeptide comprising a diphtheriatoxin T-cell epitope comprises the amino acid sequence:QSIALSSLMVAQAIPLVGEL (SEQ ID NO:117). In some cases, a suitable HCVT-cell epitope polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: VDIGFAAYNFVESIINLFQV (SEQ ID NO:118).In some cases, a suitable HCV T-cell epitope polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence:QGESGHDIKITAENTPLPIA (SEQ ID NO:119). In some cases, a suitable HCVT-cell epitope polypeptide comprising a diphtheria toxin T-cell epitopecomprises the amino acid sequence: GVLLPTIPGKLDVNKSKTHI (SEQ ID NO:120).In some cases, a suitable HCV T-cell epitope polypeptide comprising adiphtheria toxin T-cell epitope comprises the amino acid sequence ofCRM197 (see, e.g., Giannini et al. (1984) Nucl. Acids. Res. 12:4063).

The amino acid sequence of CRM197 is as follows:

(SEQ ID NO: 121) laddvvdssksfvmenfssyhgtkpgyvdsiqkgiqkpksgtqgnydddwkefystdnkydaagysvdnenplsgkaggvvkvtypgltkvlalkvdnaetikkelglslteplmeqvgteefikrfgdgasrvvlslpfaegsssveyinnweqakalsveleinfetrgkrgqdamyeymaqacagnrvrrsvgsslscinldwdvirdktktkieslkehgpiknkmsespnktvseekakqyleefhqtalehpelselktvtgtnpvfaganyaawavnvaqvidsetadnlekttaalsilpgigsvmgiadgavhhnteeivaqsialsslmvaqaiplvgelvdigfaaynfvesiinlfqvvhnsynrpayspghktqpflhdgyavswntvedsiirtgfqgesghdikitaentplpiagvllptipgkldvnkskthisvngrkirmrcraidgdvtfcrpkspvyvgngvhanlhvafhrsssekihsneissdsigvlgyqktvdhtkvnsklslffeiks.

In some cases, an HCV T-cell epitope polypeptide can comprise a tetanustoxin T-cell epitope and a diphtheria toxin T-cell epitope. In some ofthese cases, the HCV T-cell epitope polypeptide can comprise the aminoacid sequence: IMQYIKANSKFIGIQSIALSSLMVAQ (SEQ ID NO:122); and can havea length of from 26 amino acids to 30 amino acids.

Mixtures of HCV T-cell epitope polypeptides

In some cases, an immunogenic composition of the present disclosurecomprises two or more different HCV T-cell epitope polypeptidescomprising a T-cell epitope present in an HCV protein other than E1 andE2 (e.g., a mixture of two or more different HCV T-cell epitopepolypeptides comprising a T-cell epitope present in an HCV protein otherthan E1 and E2).

For example, in some cases, an immunogenic composition of the presentdisclosure comprises: a) two or more different HCV T-cell epitopepolypeptides comprising a T-cell epitope present in an HCV protein otherthan E1 and E2; and b) a pharmaceutically acceptable excipient. In somecases, an immunogenic composition of the present disclosure comprises:a) two or more different HCV T-cell epitope polypeptides comprising aT-cell epitope present in an HCV protein other than E1 and E2; and b) apharmaceutically acceptable excipient.

For example, the two or more different HCV T-cell epitope polypeptidescan include: i) a first HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP29,and having a length of from 29 amino acids to 35 amino acids; and ii) asecond HCV T-cell epitope polypeptide comprising an amino acid sequencehaving at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least98%, or 100%, amino acid sequence identity to TP50, and having a lengthof from 50 amino acids to 55 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP52,and having a length of from 52 amino acids to 60 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP70,and having a length of from 70 amino acids to 75 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP100,and having a length of from 100 amino acids to 110 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP171,and having a length of from 171 amino acids to 180 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP228,and having a length of from 228 amino acids to 235 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP553,and having a length of from 553 amino acids to 565 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP29, and having a length of from 29 amino acids to 35 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP778,and having a length of from 778 amino acids to 785 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP50, and having a length of from 50 amino acids to 55 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP52and having a length of from 52 amino acids to 60 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP50, and having a length of from 50 amino acids to 55 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP70and having a length of from 70 amino acids to 80 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP50, and having a length of from 50 amino acids to 55 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP100and having a length of from 100 amino acids to 110 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP50, and having a length of from 50 amino acids to 55 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP171and having a length of from 171 amino acids to 180 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP50, and having a length of from 50 amino acids to 55 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP228and having a length of from 228 amino acids to 240 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP50, and having a length of from 50 amino acids to 55 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP553and having a length of from 553 amino acids to 570 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP50, and having a length of from 50 amino acids to 55 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP778and having a length of from 778 amino acids to 790 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP52, and having a length of from 52 amino acids to 60 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP70and having a length of from 70 amino acids to 80 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP52, and having a length of from 52 amino acids to 60 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP100and having a length of from 100 amino acids to 110 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP52, and having a length of from 52 amino acids to 60 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP171and having a length of from 171 amino acids to 180 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP52, and having a length of from 52 amino acids to 60 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP228and having a length of from 228 amino acids to 240 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP52, and having a length of from 52 amino acids to 60 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP553and having a length of from 553 amino acids to 570 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP52, and having a length of from 52 amino acids to 60 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP778and having a length of from 778 amino acids to 790 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP70, and having a length of from 70 amino acids to 80 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP100and having a length of from 100 amino acids to 110 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP70, and having a length of from 70 amino acids to 80 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP171and having a length of from 171 amino acids to 190 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP70, and having a length of from 70 amino acids to 80 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP228and having a length of from 228 amino acids to 240 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP70, and having a length of from 70 amino acids to 80 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP553and having a length of from 553 amino acids to 570 amino acids.

As another example, the two or more different HCV T-cell epitopepolypeptides can include: i) a first HCV T-cell epitope polypeptidecomprising an amino acid sequence having at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100%, amino acid sequenceidentity to TP70, and having a length of from 70 amino acids to 80 aminoacids; and ii) a second HCV T-cell epitope polypeptide comprising anamino acid sequence having at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100%, amino acid sequence identity to TP778and having a length of from 778 amino acids to 790 amino acids.

Pharmaceutically Acceptable Excipients

As noted above, an immunogenic composition of the present disclosure cancomprise: a) an HCV T-cell epitope polypeptide; and b) apharmaceutically acceptable excipient. A wide variety ofpharmaceutically acceptable excipients is known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy”, 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds7th ed., Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. PharmaceuticalAssoc.

In some embodiments, an HCV T-cell epitope polypeptide is formulated inan aqueous buffer. Suitable aqueous buffers include, but are not limitedto, acetate, succinate, citrate, and phosphate buffers varying instrengths from about 5 mM to about 100 mM. In some embodiments, theaqueous buffer includes reagents that provide for an isotonic solution.Such reagents include, but are not limited to, sodium chloride; andsugars e.g., mannitol, dextrose, sucrose, and the like. In someembodiments, the aqueous buffer further includes a non-ionic surfactantsuch as polysorbate 20 (TWEEN®20) or polysorbate 80 (TWEEN®80). Forexample, a formulation of an HCV T-cell epitope polypeptide in anaqueous buffer can include, e.g., from about 0.01% to about 0.05%polysorbate-20 (TWEEN®20) non-ionic detergent. Optionally theformulations may further include a preservative. Suitable preservativesinclude, but are not limited to, a benzyl alcohol, phenol,chlorobutanol, benzalkonium chloride, and the like. In many cases, theformulation is stored at about 4° C. Formulations may also belyophilized, in which case they generally include cryoprotectants suchas sucrose, trehalose, lactose, maltose, mannitol, and the like.Lyophilized formulations can be stored over extended periods of time,even at ambient temperatures. In some cases, the aqueous buffer furtherincludes a non-ionic surfactant. In some cases, the aqueous bufferincludes the non-ionic surfactant Triton™X-100, e.g., 0.1% Triton™X-100.

An HCV T-cell epitope polypeptide can be formulated into a preparationfor injection by dissolving, suspending or emulsifying the polypeptidein an aqueous or nonaqueous solvent, such as vegetable or other similaroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives.

An immunogenic composition of the present disclosure can include, e.g.,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like.

The concentration of an HCV T-cell epitope polypeptide in a formulationcan vary widely (e.g., from less than about 0.1% to at least about 2%,to as much as 20% to 50% or more by weight) and can be selectedprimarily based on fluid volumes, viscosities, and patient-based factorsin accordance with the particular mode of administration selected andthe patient's needs.

An immunogenic composition of the present disclosure can be provided inthe form of a solution, suspension, tablet, pill, capsule, powder, gel,cream, lotion, ointment, aerosol or the like. It is recognized that oraladministration can require protection of the compositions fromdigestion. This is typically accomplished either by association of thecomposition with an agent that renders it resistant to acidic andenzymatic hydrolysis or by packaging the composition in an appropriatelyresistant carrier. Means of protecting from digestion are well known inthe art.

An immunogenic composition of the present disclosure can also beprovided so as to enhance serum half-life of the polypeptides (an HCVT-cell epitope polypeptide), following administration. For example,where an HCV T-cell epitope polypeptide is formulated for injection, thepolypeptide may be provided in a liposome formulation, prepared as acolloid, or other conventional techniques for extending serum half-life.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The preparations may alsobe provided in controlled release or slow-release forms.

Adjuvant

An immunogenic composition of the present disclosure can include anadjuvant. Examples of known suitable adjuvants that can be used inhumans include, but are not necessarily limited to, alum (e.g., aluminumphosphate; aluminum hydroxide), MF59™ (4.3% w/v squalene, 0.5% w/v Tween80™ (polysorbate 80), 0.5% w/v Sorbitan trioleate (Span® 85)),CpG-containing nucleic acid (where the cytosine is unmethylated), QS21,monophosphoryl lipid A (MPL), 3-Q-desacyl-4′-monophosphoryl lipid A(3DMPL), extracts from Aquilla, immune-stimulating complexes (ISCOMS;complexes of cholesterol, phospholipids, and Quillaia saponins), LT/CTmutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, saponinvaccine adjuvant (Quil A®), interleukins, and the like. For experimentalanimals, one can use Freund's incomplete adjuvant, or Freund's completeadjuvant. Also suitable for use areN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI™, which contains threecomponents extracted from bacteria: monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene Tween80™ (polysorbate 80) emulsion. The effectiveness of an adjuvant may bedetermined by one or more of measuring the amount of antibodies directedagainst the immunogenic antigen or antigenic epitope thereof, measuringa cytotoxic T lymphocyte response to the antigen, and measuring a helperT cell response to the antigen.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59™ (see, e.g., WO 90/14837), containing 5% Squalene, 0.5%Tween 80™ (polysorbate 80), and 0.5% Sorbitan trioleate (Span® 85)(optionally containing MTP-PE) formulated into submicron particles usinga microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80™(polysorbate 80), 5% pluronic-blocked polymer L121, and thr-MDP eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) RIBI™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80™ (polysorbate 80), and one or more bacterial cell wall componentssuch as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cellwall skeleton (CWS), e.g., MPL+CWS (Detox™); (2) saponin adjuvants, suchas QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.; a purifiedextract of Quillaja saponaria) may be used or particles generatedtherefrom such as ISCOMs (immunostimulating complexes), which ISCOMS maybe devoid of additional detergent e.g. WO 00/07621; (3) CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4)cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon),macrophage colony stimulating factor (M-CSF), tumor necrosis factor(TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL(3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions (see, e.g. EP-A-0835318, EP-A-0735898,EP-A-0761231); (7) oligonucleotides comprising a CpG motif containing atleast one CG dinucleotide, where the cytosine is unmethylated (see,e.g., WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495,WO 98/37919 and WO 98/52581); (8) a polyoxyethylene ether or apolyoxyethylene ester (see, e.g. WO 99/52549); (9) a polyoxyethylenesorbitan ester surfactant in combination with an octoxynol (WO 01/21207)or a polyoxyethylene alkyl ether or ester surfactant in combination withat least one additional non-ionic surfactant such as an octoxynol (WO01/21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g.a CpG oligonucleotide) (WO 00/62800); (11) an immunostimulant and aparticle of metal salt (see, e.g. WO 00/23105); (12) a saponin and anoil-in-water emulsion (see e.g. WO 99/11241); (13) a saponin (e.g.QS21)+3dMPL+IM2 (optionally including a sterol) (see, e.g. WO 98/57659);(14) other substances that act as immunostimulating agents to enhancethe efficacy of the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc. Also suitable for use is Matrix-M™; Matrix-M™ is anadjuvant that comprises 40 nm nanoparticles comprising Quillajasaponins, cholesterol, and phospholipid. Adjuvants suitable foradministration to a human are of particular interest. In some cases, theadjuvant is one that enhances a CD4⁺ T helper response to the immunogen.Also suitable for use is a poly inosine:cytosine (poly I:C) nucleicacid. Poly I:C is a synthetic double-stranded RNA Also suitable for useis a cyclic dinucleotide activator of the STING pathway. Examples ofsuitable cyclic dinucleotide adjuvants include, but are not limitedto: 1) bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP); 2)bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP); andbis-(3′,5′)-cyclic dimeric inosine monosphosphate (c-di-IMP).

In some instances, the adjuvant is MF59, with or without aCpG-containing oligonucleotide. In other instances, the adjuvant isalum, with or without a CpG-containing oligonucleotide. In otherinstances, the adjuvant is poly(D,L-lactide-co-glycolide), with orwithout a CpG-containing oligonucleotide. In other instances, theadjuvant is MPL, with or without a CpG-containing oligonucleotide. Insome cases, the adjuvant is Matrix-M™, with or without a CpG-containingoligonucleotide. In some cases, the adjuvant is keyhole limpethemocyanin. In some cases, the adjuvant is alum. In some cases, theadjuvant is aluminum phosphate. In some cases, the adjuvant is aluminumhydroxide. In some cases, the adjuvant is alum+MPL. In some cases, theadjuvant is MF59. In some cases, the adjuvant is alum+MF59. In somecases, the adjuvant is AS01. AS01 contains QS-21 Stimulon® adjuvant,MPL, and liposomes. In some cases, the adjuvant is AS03. A dose of S03contains: 10.69 mg squalene; 11.86 mg DL-a-tocopherol; and 4.86 mgpolysorbate-80. In some cases, the adjuvant is AS04. In some cases, theadjuvant is AS15. AS15 is a combination of QS-21 Stimulon® adjuvant,monophosphoryl lipid A, and CpG7909 (an oligonucleotide of the sequence5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; (SEQ ID NO:123), in a liposomalformulation.

Methods of Inducing or Enhancing an Immune Response

The present disclosure provides methods of inducing or enhancing animmune response in an individual, the method comprising administering tothe individual an effective amount of an immunogenic composition of thepresent disclosure, where the immunogenic composition comprises an HCVT-cell epitope polypeptide, and where the immunogenic composition doesnot include an HCV E1/E2 heterodimer, an HCV E1 polypeptide, or an HCVE2 polypeptide.

Methods of Making an HCV E1/E2 Heterodimer, and for Making AHeterologous Polypeptide

An HCV E1/E2 heterodimer, an HCV E2 polypeptide, and a heterologouspolypeptide, suitable for inclusion in an immunogenic composition of thepresent disclosure, can be generated using standard methods forproducing a polypeptide in a host cell.

An HCV E1/E2 heterodimer, an HCV E2 polypeptide, and a heterologouspolypeptide, suitable for inclusion in an immunogenic composition of thepresent disclosure, can be produced using any suitable method, includingrecombinant and non-recombinant methods (e.g., chemical synthesis). AnHCV E1/E2 heterodimer, an HCV E2 polypeptide, and a heterologouspolypeptide, suitable for inclusion in an immunogenic composition of thepresent disclosure, can be generated using standard methods forproducing a polypeptide in a host cell.

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as Fmoc and Boc, areavailable for synthesizing polypeptides. Details of the chemicalsynthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. MedChem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett. 12:723-8).

Where a polypeptide is produced using recombinant techniques, thepolypeptide may be produced as an intracellular protein or as ansecreted protein, using any suitable construct and any suitable hostcell, which can be a prokaryotic or eukaryotic cell, such as a bacterial(e.g., Escherichia coli) cell or a yeast host cell, respectively. Otherexamples of eukaryotic cells that may be used as host cells includeinsect cells, mammalian cells, filamentous fungi, and plant cells.Suitable yeast cells include, e.g., Saccharomyces cerevisiae and Pichia(e.g., Pichia pastoris).

In some cases, the heterologous polypeptide is produced separately from(e.g., in a separate host cell) from the HCV E1/E2 heterodimer. In somecases, the heterologous polypeptide is produced is a first host cell;and the HCV E1/E2 heterodimer is produced in a second host cell. Oncethe HCV E1/E2 heterodimer and the heterologous polypeptide areseparately produced, they can be combined, together with apharmaceutically acceptable excipient, to generate an immunogeniccomposition of the present disclosure. In some cases, both the HCV E1/E2heterodimer and the heterologous polypeptide are purified before beingcombined to generate an immunogenic composition. For example, both theHCV E1/E2 heterodimer and the heterologous polypeptide can be at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or more than 99%, pure, e.g., free from other polypeptides, othermacromolecules, etc.

Methods of Making a Heterologous Polypeptide

A heterologous polypeptide present in an immunogenic composition of thepresent disclosure can be generated using any known method for making apolypeptide. In some cases, a nucleic acid (e.g., a recombinantexpression vector) comprising a nucleotide sequence encoding theheterologous polypeptide is introduced into a host cell, generating agenetically modified (recombinant) host cell, where the recombinantexpression vector provides for expression of the heterologouspolypeptide in the genetically modified host cell.

In some cases, the heterologous polypeptide is produced as a fusionpolypeptide comprising: a) the heterologous polypeptide; and b) a fusionpartner, where the fusion partner is an affinity tag. Suitable affinitytags include, e.g., immunoglobulin Fc polypeptides, a poly(histidine)tag (e.g., His₆), a maltose binding protein (MBP), aglutathione-S-transferase (GST) polypeptide, calmodulin-binding peptide(CBP), Streptavidin-binding peptide (SBP), Strep-tag II, FLAG (e.g.,DYKDDDDK (SEQ ID NO:91), hemagglutinin (HA) (e.g., YPYDVPDYA (SEQ IDNO:92), c-myc T7 ((e.g., EQKLISEEDL; SEQ ID NO:93), Glu-Glu,starch-binding domain (SBD), and Flag-Acidic-Target Tag (FATT), and thelike.

In some cases, the heterologous polypeptide is produced as a fusionpolypeptide comprising: a) the heterologous polypeptide; and b) a fusionpartner (e.g., where the fusion partner is an Ig Fc polypeptide). Insome cases, a proteolytically cleavable linker is interposed between theheterologous polypeptide and the fusion partner, such that the fusionpolypeptide comprises: a) the heterologous polypeptide; b) theproteolytically cleavable linker; and c) the fusion partner (e.g., Ig Fcpolypeptide).

The proteolytically cleavable linker can include a protease recognitionsequence recognized by a protease selected from the group consisting ofalanine carboxypeptidase, Armillaria mellea astacin, bacterial leucylaminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosolalanyl aminopeptidase, elastase, endoproteinase Arg-C, enterokinase,gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase,human rhinovirus 3C protease, hypodermin C, IgA-specific serineendopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC,lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionylaminopeptidase, myxobacter, nardilysin, pancreatic endopeptidase E,picornain 2A, picornain 3C, proendopeptidase, prolyl aminopeptidase,proprotein convertase I, proprotein convertase II, russellysin,saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissuekallikrein, tobacco etch virus (TEV), togavirin, tryptophanylaminopeptidase, U-plasminogen activator, V8, venombin A, venombin AB,and Xaa-pro aminopeptidase.

For example, the proteolytically cleavable linker can comprise a matrixmetalloproteinase cleavage site, e.g., a cleavage site for a MMPselected from collagenase-1, -2, and -3 (MMP-1, -8, and -13), gelatinaseA and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11),matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP andMT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy(wherein, X represents an arbitrary residue; Hy, a hydrophobic residue),e.g., Pro-X-X-Hy-(Ser/Thr), e.g., Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ IDNO:125) or Pro-Leu/Gln-Gly-Met-Thr (SEQ ID NO:75). Another example of aprotease cleavage site is a plasminogen activator cleavage site, e.g., auPA or a tissue plasminogen activator (tPA) cleavage site. In somecases, the cleavage site is a furin cleavage site. Specific examples ofcleavage sequences of uPA and tPA include sequences comprisingVal-Gly-Arg. Another example of a protease cleavage site that can beincluded in a proteolytically cleavable linker is a tobacco etch virus(TEV) protease cleavage site, e.g., ENLYTQS (SEQ ID NO:126), where theprotease cleaves between the glutamine and the serine. Another exampleof a protease cleavage site that can be included in a proteolyticallycleavable linker is an enterokinase cleavage site, e.g., DDDDK (SEQ IDNO:77), where cleavage occurs after the lysine residue. Another exampleof a protease cleavage site that can be included in a proteolyticallycleavable linker is a thrombin cleavage site, e.g., LVPR (SEQ ID NO:78).Additional suitable linkers comprising protease cleavage sites includelinkers comprising one or more of the following amino acid sequences:LEVLFQGP (SEQ ID NO:65), cleaved by PreScission protease (a fusionprotein comprising human rhinovirus 3C protease andglutathione-S-transferase; Walker et al. (1994) Biotechnol. 12:601); athrombin cleavage site, e.g., CGLVPAGSGP (SEQ ID NO:79); SLLKSRMVPNFN(SEQ ID NO:80) or SLLIARRMPNFN (SEQ ID NO:82), cleaved by cathepsin B;SKLVQASASGVN (SEQ ID NO:74) or SSYLKASDAPDN (SEQ ID NO:70), cleaved byan Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO:71) cleaved byMMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO:127) cleaved by MMP-7(matrilysin); SPQGIAGQRNFN (SEQ ID NO:72) cleaved by MMP-9;DVDERDVRGFASFL SEQ ID NO:73) cleaved by a thermolysin-like MMP;SLPLGLWAPNFN (SEQ ID NO:124) cleaved by matrix metalloproteinase 2(MMP-2); SLLIFRSWANFN (SEQ ID NO:128) cleaved by cathespin L;SGVVIATVIVIT (SEQ ID NO:129) cleaved by cathepsin D; SLGPQGIWGQFN (SEQID NO:130) cleaved by matrix metalloproteinase 1 (MMP-1); KKSPGRVVGGSV(SEQ ID NO:131) cleaved by urokinase-type plasminogen activator;PQGLLGAPGILG (SEQ ID NO:132) cleaved by membrane type 1matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQID NO:133) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblastcollagenase and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO:134) cleaved bymatrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE (SEQ IDNO:135) cleaved by tissue-type plasminogen activator(tPA); SLSALLSSDIFN(SEQ ID NO:136) cleaved by human prostate-specific antigen; SLPRFKIIGGFN(SEQ ID NO:137) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ IDNO:138) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:139)cleaved by calpain (calcium activated neutral protease).

Depending on the proteolytically cleavable linker, a heterologouspolypeptide (a T-cell epitope polypeptide) can comprise, at itsN-terminus or at its C-terminus, from 1 to 6 additional amino acids thatare N-terminal or C-terminal to the cleavage site of the proteolyticallycleavable linker. The following are non-limiting examples. In somecases, a heterologous polypeptide (a T-cell epitope polypeptide) is amodified T-cell epitope polypeptide that comprises from 1 to 6additional amino acids at the N-terminus of the modified T-cell epitopepolypeptide, where the from 1 to 6 additional amino acids are Gly-Pro,Ser, Gly, or Gly-Ser. In some cases, a heterologous polypeptide (aT-cell epitope polypeptide) is a modified T-cell epitope polypeptidethat comprises from 1 to 6 additional amino acids at the C-terminus ofthe modified T-cell epitope polypeptide, where the from 1 to 6additional amino acids are LEVLFQ (SEQ ID NO:76), ENLYYFQ (SEQ IDNO:83), LVPR (SEQ ID NO:78), I(E/D)GR (SEQ ID NO:90), or DDDDK (SEQ IDNO:77).

The Fc region can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc,a human IgG4 Fc, etc. In some cases, the Fc region comprises an aminoacid sequence having at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to an amino acid sequence of an Fc region depicted in FIG.5A-5C. In some cases, the Fc region comprises an amino acid sequencehaving at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to thehuman IgG1 Fc polypeptide depicted in FIG. 5A. In some cases, the Fcregion comprises an amino acid sequence having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to the human IgG2 Fc polypeptidedepicted in FIG. 5A; e.g., the Fc region comprises an amino acidsequence having at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to amino acids 99-325 of the human IgG2 Fc polypeptide depictedin FIG. 5A. In some cases, the Fc region comprises an amino acidsequence having at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to the human IgG3 Fc polypeptide depicted in FIG. 5A; e.g., theFc region comprises an amino acid sequence having at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to amino acids 19-246 of the humanIgG3 Fc polypeptide depicted in FIG. 5A.

Suitable expression vectors include, but are not limited to, baculovirusvectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids,bacterial artificial chromosomes, viral vectors (e.g. viral vectorsbased on vaccinia virus, poliovirus, adenovirus, adeno-associated virus,SV40, herpes simplex virus, human immunodeficiency virus-basedlentivirus vectors, murine leukemia virus (MVL)-based gamma retrovirusvectors, and the like), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as Escherichia coli, mammalian cells,insect cells, or yeast cells).

Suitable host cells include eukaryotic cells, such as yeast cells,insect cells, and mammalian cells. In some cases, the host cell is acell of a mammalian cell line. Suitable mammalian cell lines includehuman cell lines, non-human primate cell lines, rodent (e.g., mouse,rat) cell lines, and the like.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302.

Suitable eukaryotic host cells include, but are not limited to, Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp.,Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactic, Candidaalbicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusariumgramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonasreinhardtii, and the like. Suitable yeast cells include, e.g.,Saccharomyces cerevisiae and Pichia (e.g., Pichia pastoris).

Suitable insect cells include, e.g., Spodoptera frugiperda cells, e.g.,Sf9 cells; Spodoptera frugiperda Sf-21 cells; Trichoplusia ni cells(e.g., Tn-368 cells; High-Five™ BTI-TN5B1-4 cells); etc.

Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, MRC4 fibroblast cells, and the like.

Methods for introduction of nucleic acids into host cells include, forexample, transformation, electroporation, conjugation, calcium phosphatemethods and the like. The method for transfer can be selected so as toprovide for stable expression of the introduced polypeptide-encodingnucleic acid. The polypeptide-encoding nucleic acid can be provided asan inheritable episomal element (e.g., a plasmid) or can be genomicallyintegrated.

In some cases, the heterologous polypeptide is produced in a geneticallymodified host cell; and the heterologous polypeptide is purified fromone or more of the cell culture medium and a cell lysate made from thegenetically modified host cell. Methods of purifying a polypeptide fromcell culture medium and/or a cell lysate are known in the art andinclude, e.g., affinity chromatography, size exclusion chromatography,

In some cases, where the heterologous polypeptide is a fusion proteincomprising the heterologous polypeptide and a fusion partner, the fusionprotein is purified on an affinity column comprising an antibodyspecific for the fusion partner, or other affinity partner that bindsthe fusion partner, immobilized on an insoluble support. In some cases,where the heterologous polypeptide is a fusion protein comprising theheterologous polypeptide and a fusion partner, and where the fusionpartner is an Ig Fc polypeptide, the fusion protein can be purified on aProtein A column (i.e., affinity chromatography using Protein Aimmobilized on an insoluble support).

In some cases, where the heterologous polypeptide is a fusion proteincomprising the heterologous polypeptide and a fusion partner, and wherethe fusion partner is an Ig Fc polypeptide, and where a proteolyticallycleavable linker is interposed between the Ig Fc and the heterologouspolypeptide, the fusion protein can be purified on a Protein A column(i.e., affinity chromatography using Protein A immobilized on aninsoluble support). The fusion protein can be immobilized on the ProteinA column; and an enzyme that cleaves a proteolytic cleavage site in theproteolytically cleavable linker is applied to the column comprising theimmobilized fusion protein; the enzyme releases the heterologouspolypeptide from the Protein A column.

Methods of Making an HCV E1/E2 Heterodimer

An HCV E1/E2 heterodimer can be produced using any suitable method,including recombinant and non-recombinant methods (e.g., chemicalsynthesis).

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as Fmoc and Boc, areavailable for synthesizing polypeptides. Details of the chemicalsynthesis are known in the art (e.g., Ganesan A. 2006 Mini Rev. MedChem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett. 12:723-8).

Where a polypeptide is produced using recombinant techniques, thepolypeptide may be produced as an intracellular protein or as ansecreted protein, using any suitable construct and any suitable hostcell, which can be a prokaryotic or eukaryotic cell, such as a bacterial(e.g., Escherichia coli) cell or a yeast host cell, respectively. Otherexamples of eukaryotic cells that may be used as host cells includeinsect cells, mammalian cells, filamentous fungi, and plant cells.Suitable yeast cells include, e.g., Saccharomyces cerevisiae and Pichia(e.g., Pichia pastoris).

Suitable mammalian cells include human cell lines, non-human primatecell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitablemammalian cell lines include, but are not limited to, HeLa cells (e.g.,American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g.,ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573),Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHKcells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells,COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No.CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2cells, MRC5 cells (ATCC No. CCL-171), and the like. Where mammalian hostcells are used, such host cells may include human cells (e.g., HeLa,293, H9 and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and C127cells); primate cells (e.g., Cos 1, Cos 7 and CV1); MRC4 cells; andhamster cells (e.g., Chinese hamster ovary (CHO) cells).

A variety of host-vector systems suitable for the expression of apolypeptide may be employed according to standard procedures known inthe art. See, e.g., Sambrook et al., 1989 Current Protocols in MolecularBiology Cold Spring Harbor Press, New York; Ausubel et al. 1995 CurrentProtocols in Molecular Biology, Eds. Wiley and Sons; “ProteinExpression: A Practical Approach” (1999) S. J. Higgins and B. D. James,eds., Oxford University Press; “Protein Expression in Mammalian Cells:Methods and Protocols (Methods in Molecular Biology)” (2012) James L.Hartley, ed., Humana Press; and “Production of Recombinant Proteins”(2005) Gerd Gellisen, ed., Wiley-VCH. Methods for introduction ofnucleic acids into host cells include, for example, transformation,electroporation, conjugation, calcium phosphate methods and the like.The method for transfer can be selected so as to provide for stableexpression of the introduced polypeptide-encoding nucleic acid. Thepolypeptide-encoding nucleic acid can be provided as an inheritableepisomal element (e.g., a plasmid) or can be genomically integrated. Avariety of appropriate vectors for use in production of a peptide ofinterest are available commercially.

Suitable expression vectors include, but are not limited to, baculovirusvectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids,bacterial artificial chromosomes, viral vectors (e.g. viral vectorsbased on vaccinia virus, poliovirus, adenovirus, adeno-associated virus,SV40, herpes simplex virus, HIV-based lentivirus vectors, murineleukemia virus (MVL)-based gamma retrovirus vectors, and the like),P1-based artificial chromosomes, yeast plasmids, yeast artificialchromosomes, and any other vectors specific for specific hosts ofinterest (such as E. coli, mammalian cells, insect cells, or yeastcells).

An E1 polypeptide, an E2 polypeptide, or an E1/E2 heterodimer can beproduced by introducing a recombinant expression vector comprising anucleotide sequence encoding the E1 polypeptide, E2 polypeptide, orE1/E2 heterodimer into an appropriate host cell, where the host cellproduces the encoded E1 polypeptide, E2 polypeptide, or E1/E2heterodimer. In the expression vector, a polynucleotide comprising anucleotide sequence(s) encoding the E1 polypeptide, E2 polypeptide, orE1/E2 heterodimer is linked to a regulatory sequence as appropriate toobtain the desired expression properties. These regulatory sequences caninclude promoters, enhancers, terminators, operators, repressors, andinducers. The promoters can be regulated or constitutive. Expressionvectors generally have convenient restriction sites located near thepromoter sequence to provide for the insertion of nucleic acid sequencesencoding a protein of interest. A selectable marker operative in theexpression host cell may be present.

In some cases, the E1/E2 heterodimer is encoded in a recombinantexpression vector suitable for expression in a eukaryotic host cell(e.g., an insect cell; a yeast cell; a mammalian host cell, such as CHOcells, HeLa cells, 293 cells, MRC5 cells, etc.). In some cases, arecombinant expression vector comprises a nucleotide sequence encodingE1 and E2 polypeptides (which may be wild-type or variant) as a singlepolypeptide chain; the recombinant expression vector is introduced intoa eukaryotic host cell to generate a genetically modified host cell. Insome cases, E1 and E2 polypeptides are initially produced as a singlepolypeptide chain, which is cleaved in the endoplasmic reticulum (ER) ofthe genetically modified host cell to produce separate E1 and E2polypeptides. The separate E1 and E2 polypeptides can form a heterodimer(e.g., a non-covalently linked heterodimer) in the ER. The E1/E2heterodimer can be isolated from the genetically modified host cell by,e.g., lysis using a non-ionic detergent, or using a freeze-thaw method.See, e.g., Frey et al. (2010) Vaccine 28:6367. The E1/E2 heterodimer canbe purified from a cell lysate and/or cell culture medium using any of avariety of methods, including size exclusion chromatography, affinitychromatography, and the like, or combinations of such methods. In somecases, the E1/E2 heterodimer is purified from cell lysate and/or cellculture medium using Galanthus nivalis (GNA) lectin affinitychromatography. In some cases, the E1/E2 heterodimer is purified from acell lysate. In some cases, the E1/E2 heterodimer is secreted from acell and is purified from the cell culture medium. Suitable methods thatcan be used for purifying an E1/E2 heterodimer are described in, e.g.,U.S. Pat. Nos. 6,121,020; 6,274,148; and Mazzocca et al. (2005) J. Biol.Chem. 280:11329. For example, in some cases, an E1/E2 heterodimer can beprepared in a method comprising cell disruption and debris removal bymicrofiltration, followed by purification using three subsequentchromatographic steps: lectin affinity chromatography, hydroxyapatitechromatography, and ion exchange chromatography.

Alternatively, the E1 and E2 polypeptides can be encoded on separaterecombinant expression vectors; and produced in a cell (e.g., the samehost cell or separate host cells) as separate polypeptides.

If full-length E1 and E2 polypeptides are expressed in a eukaryotic hostcell, the E1 and E2 polypeptides remain bound to the endoplasmicreticulum (ER) membrane as asialoglycoproteins. If the E1 and E2polypeptides have C-terminal truncations, such that the C-terminaltransmembrane regions are removed, the truncated polypeptides aresecreted and can acquire complex glycans such as sialic acid. Removal ofapproximately amino acids 660-746 of E2, or amino acids 715-746 of E2,and removal of approximately amino acids 330-383 of E1, results insecretion of E2 and E1 from a eukaryotic host cell. If E1 and E2 areco-expressed in the same eukaryotic host cell as full-lengthpolypeptides, they remain in the lumen of the ER as a heterodimer.

In some cases, an E2 polypeptide suitable for use in an E1/E2heterodimer lacks a transmembrane region. For example, in some cases, anE2 polypeptide suitable for use in an E1/E2 heterodimer, comprises aminoacids 384-659, and lacks amino acids 660-746 of a naturally-occurring E2polypeptide; and may be referred to as “E2 ectodomain polypeptide.” Forexample, in some cases, an E2 polypeptide suitable for use in an E1/E2heterodimer comprises amino acids 384-659, lacks amino acids 660-746 ofa naturally-occurring E2 polypeptide, and has a length of 276 aminoacids.

In some cases, an E1 polypeptide suitable for use in an E1/E2heterodimer lacks a transmembrane region. For example, in some cases, anE1 polypeptide suitable for use in an E1/E2 heterodimer comprises aminoacids 191-329, and lacks amino acids 330-383 of a naturally-occurring E1polypeptide; and may be referred to as an “E1 ectodomain polypeptide.”For example, in some cases, an E1 polypeptide suitable for use in anE1/E2 heterodimer comprises amino acids 191-329, lacks amino acids330-383 of a naturally-occurring E1 polypeptide, and has a length of 139amino acids.

After production in a host cell, an E1 polypeptide, an E2 polypeptide,or an E1/E2 heterodimer (e.g., as separate polypeptides or as aheterodimer) can be purified from the host cell. Methods of purificationof recombinantly produced polypeptides from a host cell are known in theart and include, e.g., detergent lysis (e.g., with a non-ionicdetergent) or freeze-thaw lysis, followed by one or more of sizeexclusion column chromatography, high performance liquid chromatography,affinity chromatography, and the like.

In some cases, an E1/E2 heterodimer suitable for inclusion in animmunogenic composition of the present disclosure is produced bypurifying an E1/E2 heterodimer on an affinity column, where the E1 or E2polypeptide comprises an Ig Fc polypeptide linked to the E1 or E2polypeptide via a proteolytically cleavable linker. For example, themethod can comprise: A) culturing a genetically modified eukaryotic hostcell that is genetically modified with a nucleic acid (e.g., recombinantexpression vector) comprising a nucleotide sequence encoding an E1/E2polypeptide that comprises, in order from N-terminus to C-terminus: a) asignal peptide that directs the E1/E2 polypeptide to the ER followingtranslation of the E1/E2 polypeptide; b) an HCV E1 polypeptide; c) an IgFc region; d) a proteolytically cleavable linker; and e) an HCV E2polypeptide); B) contacting a lysate of the cultured geneticallymodified eukaryotic host cell with a solid support comprising an Ig Fcbinding moiety, generating an immobilized heterodimer comprising the HCVE1 polypeptide and a fusion polypeptide comprising: a) the Ig Fc; b) theproteolytically cleavable linker; and c) the E2 polypeptide; C)contacting the immobilized heterodimer with an enzyme that cleaves theproteolytically cleavable linker, thereby releasing the heterodimer; andD) collecting the released heterodimer.

In some cases, an E1/E2 heterodimer of the present disclosure isproduced using a method comprising: A) culturing a genetically modifiedeukaryotic host cell that is genetically modified with a nucleic acid(e.g., recombinant expression vector) comprising a nucleotide sequenceencoding an E1/E2 polypeptide that comprises, in order from N-terminusto C-terminus: a) a signal peptide that directs the E1/E2 polypeptide tothe ER following translation of the E1/E2 polypeptide; b) an HCV E1polypeptide; c) an Ig Fc region; d) a proteolytically cleavable linker;and e) an HCV E2 polypeptide; B) contacting a lysate of the culturedgenetically modified eukaryotic host cell with a solid supportcomprising an Ig Fc binding moiety, generating an immobilizedheterodimer comprising the HCV E1 polypeptide and a fusion polypeptidecomprising: a) the Ig Fc; b) the proteolytically cleavable linker; andc) the E2 polypeptide; C) contacting the immobilized heterodimer with anenzyme that cleaves the proteolytically cleavable linker, therebyreleasing the heterodimer; and D) collecting the released heterodimer.

In some cases, an E1/E2 heterodimer of the present disclosure isproduced using a method comprising: A) culturing a genetically modifiedeukaryotic host cell that is genetically modified with a nucleic acid(e.g., recombinant expression vector) comprising a nucleotide sequenceencoding an E1/E2 polypeptide that comprises, in order from N-terminusto C-terminus: a) a signal peptide that directs the E1/E2 polypeptide tothe ER following translation of the E1/E2 polypeptide; b) an HCV E1polypeptide; c) an Ig Fc region; d) a proteolytically cleavable linkercomprising the amino acid sequence LEVLFQGP (SEQ ID NO:65), and having alength of from 8 amino acids to 15 amino acids; and e) an HCV E2polypeptide; B) contacting a lysate of the cultured genetically modifiedeukaryotic host cell with a solid support comprising an Ig Fc bindingmoiety, generating an immobilized heterodimer comprising the HCV E1polypeptide and a fusion polypeptide comprising: a) the Ig Fc; b) theproteolytically cleavable linker; and c) the E2 polypeptide; C)contacting the immobilized heterodimer with an enzyme (e.g., arhinovirus 3C protease) that cleaves the proteolytically cleavablelinker, thereby releasing the heterodimer; and D) collecting thereleased heterodimer.

In some cases, an E1/E2 heterodimer of the present disclosure isproduced using a method comprising: A) culturing a genetically modifiedeukaryotic host cell that is genetically modified with a nucleic acid(e.g., recombinant expression vector) comprising a nucleotide sequenceencoding an E1/E2 polypeptide that comprises, in order from N-terminusto C-terminus: a) a signal peptide that directs the E1/E2 polypeptide tothe ER following translation of the E1/E2 polypeptide; b) an HCV E1polypeptide; c) an Ig Fc region; d) a proteolytically cleavable linkercomprising the amino acid sequence LEVLFQGP (SEQ ID NO:65), and having alength of from 8 amino acids to 15 amino acids; and e) an HCV E2polypeptide; B) contacting a lysate of the cultured genetically modifiedeukaryotic host cell with a solid support comprising an Ig Fc bindingmoiety, generating an immobilized heterodimer comprising the HCV E1polypeptide and a fusion polypeptide comprising: a) the Ig Fc; b) theproteolytically cleavable linker; and c) the E2 polypeptide; C)contacting the immobilized heterodimer with an enzyme (e.g., a fusionpolypeptide comprising a glutathione-S-transferase and a humanrhinovirus 3C protease (GST-HRV3C protease)) that cleaves theproteolytically cleavable linker, thereby releasing the E1E2heterodimer; and D) collecting the released E1E2 heterodimer. In somecases, a solution comprising the released E1E2 heterodimer is applied toglutathione immobilized on a solid support, to remove the GST-HRV3Cprotease. For example, a solution comprising the released heterodimercan be applied to a glutathione-Sepharose 4B column, where the GST-HRV3Cbinds to the glutathione-Sepharose 4B; the flow-through (unboundmaterial) comprises the released E1E2 heterodimer. In some cases, thereleased E1E2 heterodimer is further subjected to hydroxyapatitechromatography. Hydroxyapatite chromatography can be carried out asdescribed in, e.g., Mazzocca et al. (2005) J. Biol. Chem. 280:11329.

Suitable Ig Fc binding moieties include, but are not limited to, ProteinA (Graille et al. (2000) Proc. Natl. Acad. Sci. USA 97:5399); Protein G(Sjobring et al. (1991) J. Biol. Chem. 266:399); and a Protein A/Gfusion polypeptide (Eliasson et al. (1988) J. Biol. Chem. 263:4323).

The Ig Fc binding moiety can be immobilized onto a solid support, wherethe solid support can be of any of a variety of forms, e.g., a bead, amagnetic bead, a plate, and the like. The solid support can be made ofany of a variety of materials, including, but not limited to,polystyrene, agarose, polyesters, polyethylene, and the like.

As an alternative to Fc, an affinity tag such as, e.g., polyhistidine(e.g., (His)₆), glutathione-S-transferase (GST), calmodulin-bindingpeptide (CBP), Streptavidin-binding peptide (SBP), Strep-tag II, FLAG(e.g., DYKDDDDK (SEQ ID NO:91), hemagglutinin (HA) (e.g., YPYDVPDYA (SEQID NO:92), c-myc T7 ((e.g., EQKLISEEDL; SEQ ID NO:93), Glu-Glu, and thelike, can be used. (Wood D. 2014. Current Opinion in Structural Biology26 54-61; Kimple M E et al. 2013. Current Protocols in Protein Science9.9.1-9.9.23). Other suitable affinity tags include, e.g.,starch-binding domain (SBD); and Flag-Acidic-Target Tag (FATT). See,e.g., Wood D. 2014. Current Opinion in Structural Biology 26 54-61).

One or more additional purification steps can be carried out. Forexample, a solution comprising the released heterodimer, produced asdescribed above, can be subjected to size exclusion chromatography,hydroxyapatite chromatography, and the like. Hydroxyapatitechromatography can be carried out as described in, e.g., Mazzocca et al.(2005) J. Biol. Chem. 280:11329.

An E1/E2 heterodimer of the present disclosure can be purified such thatthe E1/E2 heterodimer is at least 60% pure, at least 65% pure, at least70% pure, at least 75% pure, at least 80% pure, at least 85% pure, atleast 90% pure, at least 95% pure, at least 98% pure, at least 99% pure,or greater than 99% pure.

Nucleic Acid Immunogenic Compositions

The present disclosure provides nucleic acid compositions comprising: a)one or more nucleic acids comprising a nucleotide sequence(s) encodingpolypeptides (e.g., HCV E1/E2; HCV E1; HCV E2; T-cell epitopepolypeptide) as described above. The present disclosure provides animmunogenic composition comprising: a) a nucleic acid (e.g., arecombinant viral expression vector(s)) comprising nucleotidesequence(s) encoding one or more of: an HCV E1/E2 heterodimer; an HCV E1polypeptide; an HCV E2 polypeptide; and a T-cell epitope polypeptide,where such polypeptides are described above. The polypeptides can beencoded in the same nucleic acid, or on separate nucleic acids. Forexample, where the nucleic acid(s) are recombinant expression vectors,the polypeptides can be encoded in the same or separate recombinantexpression vectors.

In some cases, the nucleic acid(s) is/are DNA. In some cases, thenucleic acid(s) is/are RNA. In some cases, the nucleic acid(s) is/arepresent in expression vector(s), generating recombinant expressionvector(s) comprising the nucleic acid(s). In some cases, the recombinantexpression vector(s) is/are recombinant bacterial vectors. In somecases, the recombinant expression vector(s) is/are recombinant viralvector(s). In some cases, the recombinant viral vector(s) are packagedinto viral particles. In some cases, the nucleic acid(s) are present inbacteria (e.g., non-pathogenic bacteria (e.g., attenuated bacteria)suitable for delivery of nucleic acids to an individual). Where therecombinant expression vector is a bacterial vector or a viral vector,the vector is suitably attenuated so as not to cause significantpathology in an individual.

In some cases, the nucleic acid is present in an expression vector.Suitable expression vectors include, but are not limited to, areplication-defective adenovirus vector; a replication-defectivevaccinia virus vector; a lentivirus vector (e.g., a self-inactivatinglentivirus vector); a retroviral vector (e.g., a self-inactivatingretroviral vector); an adeno-associated virus vector; and the like. Insome cases, the vector is a modified vaccinia Ankara (MVA) vector, or anMVA-based vector (see, e.g., Verheust et al. (2012) Vaccine 30:2623). Insome cases, the vector is a replication-defective adenovirus vector. Insome cases, the vector is a replication-defective adenovirus 6 (Ad6)vector. In some cases, the vector is a replication-defective simianadenovirus vector (e.g., ChAd3). Suitable viral vectors are describedin, e.g., Thou et al. (2012) Invest. Ophthalmol. Vis. Sci. 53:2804;Swadling et al. (2014) Sci. Transl. Med. 6:261ra153; and Choi and Chang(2013) Clin. Exp. Vaccine Res. 2:97. In many cases, the recombinantviral vectors are packaged into viral particles; and the viral particlesare formulated in an immunogenic composition along with apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers are described above.

In some cases, an immunogenic composition of the present disclosurecomprises: a) a recombinant viral vector comprising nucleotide sequencesencoding one or more of: an HCV E1/E2 heterodimer; an HCV E1polypeptide; an HCV E2 polypeptide; and a T-cell epitope polypeptide;and b) a pharmaceutically acceptable excipient. In some cases, animmunogenic composition of the present disclosure comprises: a) arecombinant viral vector comprising nucleotide sequences encoding: anHCV E1/E2 heterodimer; and a T-cell epitope polypeptide; and b) apharmaceutically acceptable excipient. In some cases, an immunogeniccomposition of the present disclosure comprises: a) a recombinant viralvector comprising nucleotide sequences encoding: an HCV E1 polypeptide;and a T-cell epitope polypeptide; and b) a pharmaceutically acceptableexcipient. In some cases, an immunogenic composition of the presentdisclosure comprises: a) a recombinant viral vector comprisingnucleotide sequences encoding: an HCV E2 polypeptide; and a T-cellepitope polypeptide; and b) a pharmaceutically acceptable excipient.

In some cases, the present disclosure provides: a) a first immunogeniccomposition comprising: i) a recombinant viral vector comprisingnucleotide sequences encoding one or more of: an HCV E1/E2 heterodimer;an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cell epitopepolypeptide; and ii) a pharmaceutically acceptable excipient; and b) asecond immunogenic composition comprising: i) a recombinant viral vectorcomprising nucleotide sequences encoding one or more of: an HCV E1/E2heterodimer; an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cellepitope polypeptide; and ii) a pharmaceutically acceptable excipient.

In some cases, the present disclosure provides: a) a first immunogeniccomposition comprising: i) a first recombinant viral vector comprisingnucleotide sequences encoding one or more of: an HCV E1/E2 heterodimer;an HCV E1 polypeptide; an HCV E2 polypeptide; and a T-cell epitopepolypeptide; and ii) a pharmaceutically acceptable excipient; and b) asecond immunogenic composition comprising: i) a second recombinant viralvector comprising nucleotide sequences encoding one or more of: an HCVE1/E2 heterodimer; an HCV E1 polypeptide; an HCV E2 polypeptide; and aT-cell epitope polypeptide; and ii) a pharmaceutically acceptableexcipient. In some cases, the first recombinant viral vector is areplication-defective adenovirus-based recombinant viral vector; and thesecond recombinant viral vector is an MVA-based recombinant viralvector. In some cases, the first recombinant viral vector is achimpanzee adenovirus-based recombinant viral vector; and the secondrecombinant viral vector is an MVA-based recombinant viral vector.

In some cases, the nucleic acid(s) are present in bacteria (e.g.,non-pathogenic bacteria suitable for delivery of nucleic acids to anindividual). In some cases, the nucleic acid(s) are present inrecombinant expression vector(s) present in bacteria (e.g.,non-pathogenic bacteria suitable for delivery of nucleic acids to anindividual). Thus, the present disclosure provides an immunogeniccomposition comprising a non-pathogenic, bacterium that harbors anucleic acid(s) comprising nucleotide sequences encoding one or more of:an HCV E1/E2 heterodimer; an HCV E1 polypeptide; an HCV E2 polypeptide;and a T-cell epitope polypeptide, where such polypeptides are describedabove. The present disclosure provides an immunogenic compositioncomprising a non-pathogenic bacterium that harbors a recombinantexpression vector(s) comprising nucleotide sequences encoding one ormore of: an HCV E1/E2 heterodimer; an HCV E1 polypeptide; an HCV E2polypeptide; and a T-cell epitope polypeptide, where such polypeptidesare described above. In some cases, the bacteria are live. In somecases, the bacteria are live attenuated bacteria. In some cases, thebacteria are killed.

Bacteria suitable for delivery of nucleic acid(s) (which nucleic acid(s)may be present in expression vector(s)) include, but are not limited to,Lactobacillus; Lactococcus (e.g., Lactococcus lactic); Salmonella, e.g.,attenuated, non-pathogenic Salmonella, e.g., Salmonella enterica serovarTyphi, Salmonella enterica serovar Typhimurium; non-pathogenic strainsof Francisella; non-pathogenic strains of Escherichia coli;non-pathogenic strains of Bordetella pertussis; non-pathogenic strainsof Listeria; non-pathogenic strains of Shigella; non-pathogenic strainsof Vibrio (e.g., Vibrio cholera); Streptococcus gordonii; non-pathogenicstrains of Yersinia enterocolitica; non-pathogenic strains of Shigellaflexneri; non-pathogenic strains of Pseudomonas aeruginosa;non-pathogenic strains of Bacillus subtilis; and the like.

In some cases, one or more virulence genes in the bacterium is all orpartially deleted. For example, for Salmonella enterica serovar Typhiand Salmonella enterica serovar Typhimurium, an aroA, aroC, and aroDmutation can be made. Other mutations that can attenuate pathogenicityaffect biosynthesis of the nucleotides adenine (pur) and guanine(guaBA), and outer membrane proteins C and F (ompC, ompF), as well asexpression of the cAMP receptor (cya/crp), the conversion ofUDP-galactose to UDP-glucose (galE), DNA recombination and repair (recA,recBC), and regulation of virulence genes (phoP, phoQ). For Listeriamonocytogenes, attenuation can be achieved with auxotrophic mutants, ordeletion of virulence factors such as the genes actA and internalin B(intB).

Methods of Inducing an Immune Response to HCV

The present disclosure provides a method of inducing an immune response(e.g., a protective immune response) to at least one HCV genotype in amammalian subject. In some cases, a method of the present disclosure forinducing an immune response in an individual to at least one HCVgenotype comprises administering an immunogenic composition of thepresent disclosure, where the immunogenic composition comprisespolypeptides (e.g., HCV E1/E2; HCV E1; HCV E2; T-cell epitopepolypeptide). In some cases, a method of the present disclosure forinducing an immune response in an individual to at least one HCVgenotype comprises administering an immunogenic composition of thepresent disclosure, where the immunogenic composition comprises one ormore nucleic acids comprising nucleotide sequences encoding polypeptides(e.g., HCV E1/E2; HCV E1; HCV E2; T-cell epitope polypeptide).

Administering an Immunogenic Composition Comprising Polypeptides

In some cases, the methods comprise administering to an individual inneed thereof an effective amount of an immunogenic composition of thepresent disclosure, where the immunogenic composition comprises: a) anHCV E1/E2 heterodimer; b) a T-cell epitope polypeptide comprising aT-cell epitope present in an HCV polypeptide other than E1 and E2; andc) a pharmaceutically acceptable excipient; or where the immunogeniccomposition comprises: a) an HCV E2 polypeptide; b) a T-cell epitopepolypeptide comprising a T-cell epitope present in an HCV polypeptideother than E1 and E2; and c) a pharmaceutically acceptable excipient; orwhere the immunogenic composition comprises: a) an HCV E1 polypeptide;b) a T-cell epitope polypeptide comprising a T-cell epitope present inan HCV polypeptide other than E1 and E2; and c) a pharmaceuticallyacceptable excipient.

Administering an Immunogenic Composition Comprising Nucleic Acid(s)

In some cases, a method of the present disclosure for inducing an immuneresponse to HCV in an individual comprises administering to theindividual an effective amount of a nucleic acid(s) comprisingnucleotide sequences encoding: 1) an HCV E1/E2 heterodimer and a T-cellepitope polypeptide; 2) an HCV E2 polypeptide and a T-cell epitopepolypeptide; or 3) an HCV E1 polypeptide and a T-cell epitopepolypeptide. The polypeptides can be encoded in the same nucleic acid,or on separate nucleic acids. For example, where the nucleic acid(s) arerecombinant expression vectors, the polypeptides can be encoded in thesame or separate recombinant expression vectors.

In some cases, the nucleic acid(s) is/are DNA. In some cases, thenucleic acid(s) is/are RNA. In some cases, the nucleic acid(s) is/arepresent in expression vector(s) such that a recombinant expressionvector(s) comprising the nucleic acid(s) are administered. In somecases, the recombinant expression vector(s) is/are recombinant viralvector(s). In some cases, the recombinant viral vector(s) are packagedinto viral particles. In some cases, the nucleic acid(s) are present inbacteria (e.g., non-pathogenic bacteria (e.g., attenuated bacteria)suitable for delivery of nucleic acids to an individual).

In some cases, the nucleic acid is present in an expression vector,thereby generating a recombinant expression vector. Suitable expressionvectors include, but are not limited to, a replication-defectiveadenovirus vector; a replication-defective vaccinia virus vector; alentivirus vector (e.g., a self-inactivating lentivirus vector); aretroviral vector (e.g., a self-inactivating retroviral vector); anadeno-associated virus vector; and the like. In some cases, the vectoris a modified vaccinia Ankara (MVA) vector, or an MVA-based vector (see,e.g., Verheust et al. (2012) Vaccine 30:2623). In some cases, the vectoris a replication-defective adenovirus vector. In some cases, the vectoris a replication-defective adenovirus 6 (Ad6) vector. In some cases, thevector is a replication-defective simian adenovirus vector (e.g.,ChAd3). Suitable viral vectors are described in, e.g., Thou et al.(2012) Invest. Ophthalmol. Vis. Sci. 53:2804; Swadling et al. (2014)Sci. Transl. Med. 6:261ra153; and Choi and Chang (2013) Clin. Exp.Vaccine Res. 2:97. In many cases, the recombinant viral vectors arepackaged into viral particles; and the viral particles are formulated inan immunogenic composition along with a pharmaceutically acceptablecarrier.

In some cases, an HCV E1/E2 heterodimer is encoded by nucleotidesequences present in a first recombinant viral vector, e.g., anadenovirus vector, a vaccinia virus vector, an MVA vector or MVA-basedvector; and a T-cell epitope polypeptide is encoded by nucleotidesequences present in a second recombinant viral vector, e.g., anadenovirus vector, a vaccinia virus vector, an MVA vector or MVA-basedvector.

In some cases, a prime-boost vaccine protocol is used. In some cases, afirst (priming) immunogenic composition is administered, where the firstimmunogenic composition comprises a recombinant viral vector comprisingnucleotide sequences encoding one or more of: a) an HCV E1/E2heterodimer; b) an HCV E2 polypeptide; c) an HCV E1 polypeptide; and d)a T-cell epitope polypeptide; and, after a time, a second (booster)immunogenic composition is administered, where the second immunogeniccomposition comprises a recombinant viral vector comprising nucleotidesequences encoding one or more of: a) an HCV E1/E2 heterodimer; b) anHCV E2 polypeptide; c) an HCV E1 polypeptide; and d) a T-cell epitopepolypeptide. In some cases, the first recombinant viral vector and thesecond recombinant viral vector are the same. In some cases, the firstrecombinant viral vector and the second recombinant viral vector aredifferent. For example, in some cases, the first recombinant viralvector is a vaccinia-based recombinant viral vector; and the secondrecombinant viral vector is an adenovirus-based recombinant viralvector. In general, the recombinant viral vectors are packaged intoviral particles. A second immunogenic composition can be administered ata time period of from 1 day to 1 year following administration of thefirst immunogenic composition. For example, a second immunogeniccomposition can be administered at a time period of from 1 day to 1week, from 1 week to 2 weeks, from 2 weeks to 1 month, from 1 month to 2months, from 2 months to 6 months, or from 6 months to 1 year followingadministration of the first immunogenic composition.

For example, in some cases, a first (priming) vaccine comprising arecombinant adenovirus (e.g., Ad6 or chimpanzee Ad (e.g., ChAd3)) thatcomprises a nucleotide sequence encoding an HCV E1/E2 heterodimer isfollowed by a second (booster) vaccine comprising a recombinant MVAvector that comprises a nucleotide sequence encoding a T-cell epitopepolypeptide. Other prime-boost protocols can be used. For example,multiple primes and/or multiple boosts can be administered.

In some cases, a first (priming) immunogenic composition isadministered, where the first immunogenic composition comprises one ormore of: a) an HCV E1/E2 heterodimer; b) an HCV E2 polypeptide; c) anHCV E1 polypeptide; and d) a T-cell epitope polypeptide, as describedabove; and a second (boosting) immunogenic composition is administered,where the second immunogenic composition comprises a recombinant viralvector comprising nucleotide sequence(s) encoding one or more of: a) anHCV E1/E2 heterodimer; b) an HCV E2 polypeptide; c) an HCV E1polypeptide; and d) a T-cell epitope polypeptide, as described above.

In some cases, a first (priming) immunogenic composition isadministered, where the first immunogenic composition comprises arecombinant viral vector comprising nucleotide sequence(s) encoding oneor more of: a) an HCV E1/E2 heterodimer; b) an HCV E2 polypeptide; c) anHCV E1 polypeptide; and d) a T-cell epitope polypeptide, as describedabove; and a second (boosting) immunogenic composition is administered,where the second immunogenic composition comprises one or more of: a) anHCV E1/E2 heterodimer; b) an HCV E2 polypeptide; c) an HCV E1polypeptide; and d) a T-cell epitope polypeptide, as described above.

In some cases, a co-immunization regimen is carried out, in which apolypeptide(s) per se is administered substantially concomitantly with anucleic acid(s) encoding the polypeptide(s). For example, in some cases,a method of the present disclosure for inducing an immune response to anHCV polypeptide can comprise administering: a) a first immunogeniccomposition of the present disclosure, as described above, where theimmunogenic composition comprises: i) an HCV E1/E2 heterodimer; ii) aT-cell epitope polypeptide; and iii) a pharmaceutically acceptablecarrier; or i) an HCV E1 polypeptide; ii) a T-cell epitope polypeptide;and iii) a pharmaceutically acceptable carrier; or i) an HCV E2polypeptide; ii) a T-cell epitope polypeptide; and iii) apharmaceutically acceptable carrier; and b) a second immunogeniccomposition of the present disclosure, as described above, where theimmunogenic composition comprises: i) one or more nucleic acidscomprising nucleotide sequence encoding one or more of: an HCV E1/E2heterodimer, an HCV E1 polypeptide, an HCV E2 polypeptide, and a T-cellepitope polypeptide; and ii) a pharmaceutically acceptable carrier. Insome cases, the first and the second immunogenic compositions are in asingle formulation. In some cases, the first and the second immunogeniccompositions are in separate formulations. In some cases, the first andthe second immunogenic compositions are administered via the same routeof administration. In some cases, the first and the second immunogeniccompositions are administered via different routes of administration. Insome cases, the first and the second immunogenic compositions are inseparate formulations that are administered substantiallysimultaneously, e.g., within 1 minute, within 1 minute to 5 minutes,within 5 minutes to 15 minutes, or within 15 minutes to 30 minutes, ofone another. In some cases, the first and the second immunogeniccompositions are administered multiple times to an individual.

In some cases, a co-immunization regimen is carried out, in which apolypeptide(s) per se is administered substantially concomitantly with anucleic acid(s) encoding the polypeptide(s). For example, in some cases,a method of the present disclosure for inducing an immune response to anHCV polypeptide can comprise administering: a) a first immunogeniccomposition of the present disclosure, as described above, where theimmunogenic composition comprises: i) an HCV E1/E2 heterodimer; ii) aT-cell epitope polypeptide; and iii) a pharmaceutically acceptablecarrier; and b) a second immunogenic composition of the presentdisclosure, as described above, where the immunogenic compositioncomprises: i) one or more nucleic acids comprising nucleotide sequencesencoding an HCV E1/E2 heterodimer and a T-cell epitope polypeptide; andii) a pharmaceutically acceptable carrier. In some cases, the first andthe second immunogenic compositions are in a single formulation. In somecases, the first and the second immunogenic compositions are in separateformulations. In some cases, the first and the second immunogeniccompositions are administered via the same route of administration. Insome cases, the first and the second immunogenic compositions areadministered via different routes of administration. In some cases, thefirst and the second immunogenic compositions are in separateformulations that are administered substantially simultaneously, e.g.,within 1 minute, within 1 minute to 5 minutes, within 5 minutes to 15minutes, or within 15 minutes to 30 minutes, of one another. In somecases, the first and the second immunogenic compositions areadministered multiple times to an individual. In some cases, the one ormore nucleic acids are recombinant viral vectors.

In some cases, a method of the present disclosure for inducing an immuneresponse to HCV in an individual comprises administering to theindividual an effective amount of a nucleic acid(s) comprisingnucleotide sequences encoding 1) an HCV E1/E2 heterodimer and a T-cellepitope polypeptide; 2) an HCV E2 polypeptide and a T-cell epitopepolypeptide; or 3) an HCV E1 polypeptide and a T-cell epitopepolypeptide. In some cases, the nucleic acid is an RNA comprisingnucleotide sequences encoding a polypeptide of the present disclosure(e.g., an HCV E1/E2 heterodimer; an HCV E1 polypeptide; an HCV E2polypeptide; a T-cell epitope polypeptide, as described herein. See,e.g., Weiner (2013) Molec. Therapy 21:506; and Ulmer et al. (2012)Vaccine 30:4414. In some cases, an RNA (e.g., a single mRNA molecule; or2 mRNA molecules; or 3 mRNA molecules) comprising nucleotide sequencesencoding a polypeptide of the present disclosure is formulated with aliposome. In some cases, an RNA (e.g., a single mRNA molecule; or 2 mRNAmolecules) comprising nucleotide sequences encoding a polypeptide of thepresent disclosure is complexed with protamine. In some cases, an RNA(e.g., a single mRNA molecule; or 2 mRNA molecules) comprisingnucleotide sequences encoding a polypeptide of the present disclosure iscomplexed with1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOTAP/DOPE).

In some cases, the nucleic acid(s) are present in bacteria (e.g.,non-pathogenic bacteria suitable for delivery of nucleic acids to anindividual). In some cases, the nucleic acid(s) are present inrecombinant expression vector(s) present in bacteria (e.g.,non-pathogenic bacteria suitable for delivery of nucleic acids to anindividual). In some cases, the bacteria are live. In some cases, thebacteria are live attenuated bacteria. In some cases, the bacteria arekilled. Bacteria suitable for delivery of nucleic acid(s) (which may bepresent in expression vectors) include, but are not limited to,Lactobacillus; Lactococcus (e.g., Lactococcus lactic); Salmonella, e.g.,attenuated, non-pathogenic Salmonella, e.g., Salmonella enterica serovarTyphi, Salmonella enterica serovar Typhimurium; non-pathogenic strainsof Escherichia coli; non-pathogenic strains of Bordetella pertussis;non-pathogenic strains of Listeria; non-pathogenic strains of Shigella;non-pathogenic strains of Vibrio (e.g., Vibrio cholera); Streptococcusgordonii; non-pathogenic strains of Yersinia enterocolitica;non-pathogenic strains of Shigella flexneri; non-pathogenic strains ofPseudomonas aeruginosa; non-pathogenic strains of Bacillus subtilis; andthe like. In some cases, one or more virulence genes in the bacterium isall or partially deleted. For example, for Salmonella enterica serovarTyphi and Salmonella enterica serovar Typhimurium, an aroA, aroC, andaroD mutation can be made. Other mutations that can attenuatepathogenicity affect biosynthesis of the nucleotides adenine (pur) andguanine (guaBA), and outer membrane proteins C and F (ompC, ompF), aswell as expression of the cAMP receptor (cya/crp), the conversion ofUDP-galactose to UDP-glucose (galE), DNA recombination and repair (recA,recBC), and regulation of virulence genes (phoP, phoQ). For Listeriamonocytogenes, attenuation can be achieved with auxotrophic mutants, ordeletion of virulence factors such as the genes actA and internalin B(intB).

General Considerations

An immunogenic composition of the present disclosure is generallyadministered to a human subject who has an HCV infection or who is atrisk of acquiring an HCV infection (e.g., is at greater risk than thegeneral population of acquiring an HCV infection) so as to prevent or atleast partially arrest the development of disease and its complications.An amount adequate to accomplish this is defined as a “therapeuticallyeffective dose” or a “therapeutically effective amount.” “Prophylactic”use of a subject immunogenic composition generally refers toadministration to an individual who has not been infected with HCV.“Therapeutic” use of a subject immunogenic composition can refer to“prophylactic” use (administration to an individual who has not beeninfected with HCV) and/or to administration to an individual who has anHCV infection. A “therapeutically effective amount” of an immunogeniccomposition of the present disclosure, can be an amount that, whenadministered in one or more doses to an individual who is not infectedwith HCV, is effective to induce an immune response in the individual toHCV. A “therapeutically effective amount” of an immunogenic compositionof the present disclosure, can be an amount that, when administered inone or more doses to an individual who is infected with HCV, iseffective to enhance an immune response in the individual to HCV.

Amounts effective for therapeutic use will depend on, e.g., the mannerof administration, the weight and general state of health of thepatient, and the judgment of the prescribing physician. Single ormultiple doses of a subject immunogenic composition can be administereddepending on the dosage and frequency required and tolerated by thepatient, and route of administration.

In some cases, an effective amount of an immunogenic composition of thepresent disclosure is an amount that, when administered to an individualin one or more doses, is effective to induce an antibody response (e.g.,a neutralizing antibody response) to HCV in the individual. For example,antibody to HCV (e.g., extracellular HCV), and/or to an HCV-infectedcell, can be induced.

An effective amount of an immunogenic composition of the presentdisclosure can be an amount that, when administered to an individual inone or more doses, is effective to induce a neutralizing antibodyresponse to HCV of a variety of genotypes (e.g., genotype 1; genotype 3;etc.). A neutralizing antibody response reduces binding of HCV to one ormore host receptors for HCV and inhibits entry of HCV into a cell.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an immunogenic composition of the present disclosure is anamount that, when administered to an individual in one or more doses, iseffective to induce a cytotoxic T lymphocyte (CTL) response to HCV. Forexample, a CTL response to an HCV-infected cell can be induced.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an immunogenic composition of the present disclosure is anamount that, when administered to an individual in one or more doses, iseffective to induce a helper T lymphocyte (e.g., CD4⁺ T cell) to HCV inan individual.

In some cases, an effective amount (e.g., a therapeutically effectiveamount) of an immunogenic composition of the present disclosure is anamount that, when administered to an individual in one or more doses, iseffective to induce an antibody response (e.g., a neutralizing antibodyresponse) and/or a CTL response and/or a helper T cell response to HCVgenotype 1. In some cases, an effective amount (e.g., a therapeuticallyeffective amount) of an immunogenic composition of the presentdisclosure is an amount that, when administered to an individual in oneor more doses, is effective to induce an antibody response (e.g., aneutralizing antibody response) and/or a CTL response and/or a helper Tcell response to HCV genotype 3. In some cases, an effective amount(e.g., a therapeutically effective amount) of an immunogenic compositionof the present disclosure is an amount that, when administered to anindividual in one or more doses, is effective to induce an antibodyresponse (e.g., a neutralizing antibody response) and/or a CTL responseand/or a helper T cell response to HCV genotype 1 and HCV genotype 3. Insome cases, an effective amount (e.g., a therapeutically effectiveamount) of an immunogenic composition of the present disclosure is anamount that, when administered to an individual in one or more doses, iseffective to induce an antibody response (e.g., a neutralizing antibodyresponse) and/or a CTL response and/or a helper T cell response to HCVof any genotype.

An immunogenic composition of the present disclosure is generallyadministered in an amount effective to elicit an immune response, e.g.,a humoral immune response (e.g., an antibody response) and/or a CTLresponse, in the mammalian subject. Effective amounts for immunizationwill vary, and can generally range from about 1 μg to 100 jug per 70 kgpatient, e.g., from about 5 μg/70 kg to about 50 μg/70 kg. Substantiallyhigher dosages (e.g. 10 mg to 100 mg or more) may be suitable in oral,nasal, or topical administration routes. The initial administration canbe followed by booster immunization of the same immunogenic compositionor a different immunogenic composition. In some instances, a subjectmethod of inducing an immune response involves an initial administrationof an immunogenic composition of the present disclosure, followed by atleast one booster, and in some instances involves two or more (e.g.,three, four, or five) boosters. The interval between an initialadministration and a booster, or between a give booster and a subsequentbooster, can be from about 1 week to about 12 weeks, e.g., from about 1week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4weeks to about 6 weeks, from about 6 weeks to about 8 weeks, from about8 weeks to about 10 weeks, or from about 10 weeks to about 12 weeks. Theinterval between an initial administration and a booster, or between agive booster and a subsequent booster, can be from 4 months to 6 months,or from 6 months to 1 year.

In general, immunization can be accomplished by administration of animmunogenic composition of the present disclosure by any suitable route,including administration of the composition orally, nasally,nasopharyngeally, parenterally, enterically, gastrically, topically,transdermally, subcutaneously, intramuscularly, in tablet, solid,powdered, liquid, aerosol form, locally or systemically, with or withoutadded excipients. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980). In some instances, immunization is accomplished byintramuscular injection of an immunogenic composition of the presentdisclosure.

Individuals Suitable for Administration

Individuals who are suitable for administration with an immunogeniccomposition of the present disclosure include immunologically naïveindividuals (e.g., individuals who have not been infected with HCVand/or who have not been administered with an HCV vaccine). Individualssuitable for administration include humans.

Individuals who are suitable for administration with an immunogeniccomposition of the present disclosure composition of the presentdisclosure include individuals who are at greater risk than the generalpopulation of becoming infected with HCV, where such individualsinclude, e.g., intravenous drug users; individuals who are therecipients, or the prospective recipients, of blood or blood productsfrom another (donor) individual(s); individuals who are the recipients,or the prospective recipients, of non-autologous cells, tissues, ororgans from another (donor) individual; health care workers; emergencymedical and non-medical personnel (e.g., first responders; firefighters; emergency medical team personnel; etc.) and the like.

Individuals who are suitable for administration with an immunogeniccomposition of the present disclosure composition of the presentdisclosure include individuals who recently became exposed to HCV or whorecently became infected with HCV. For example, a subject immunogeniccomposition can be administered to an individual within from about 24hours to about 48 hours, from about 48 hours to about 1 week, or fromabout 1 week to about 4 weeks, following possible or suspected exposureto HCV or following infection with HCV.

Individuals who are suitable for administration with an immunogeniccomposition of the present disclosure composition of the presentdisclosure include individuals who have been diagnosed as having an HCVinfection, and include chronically infected individuals. In some cases,an individual who has been diagnosed as having an HCV infection istreated with an anti-viral agent and an immunogenic composition of thepresent disclosure. Suitable anti-viral agents for treating HCVinfection include, e.g., ribavirin(1-(3-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide); interferon-alpha(IFN-α) (where “IFN-α” includes IFN-α2a; IFN-α2b; IFN-α that isconjugated with poly(ethylene glycol) (“pegylated IFN-α), where thepegylated IFN-α can be pegylated IFN-α2a or pegylated IFN-α 2b); an HCVNS3 protease inhibitor (e.g., boceprevir; telaprevir); and an HCV NS5protease inhibitor. In some cases, an individual who has been diagnosedas having an HCV infection is treated with, e.g.: 1) IFN-α+ribavirin;and an immunogenic composition of the present disclosure; or 2)IFN-α+ribavirin+an HCV protease inhibitor (e.g., boceprevir ortelaprevir); and an immunogenic composition of the present disclosure.Suitable anti-viral agents for treating HCV infection include Sovaldi(Sofosbuvir; a nucleotide analog that functions as an NS5B polymeraseinhibitor), alone or in combination with pegylated IFN-α and ribavirin.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-122 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

Aspect 1. An immunogenic composition comprising: a) a hepatitis C virus(HCV) heterodimeric polypeptide comprising: i) an HCV E1 polypeptide;and ii) an HCV E2 polypeptide; b) a T-cell epitope polypeptidecomprising a T-cell epitope present in an HCV protein other than E1 andE2; and c) a pharmaceutically acceptable carrier.

Aspect 2. The immunogenic composition of aspect 1, wherein the T-cellepitope polypeptide comprises one or more T cell epitopes present in oneor more of: a) an HCV non-structural polypeptide-3 (NS3) polypeptide; b)an HCV non-structural polypeptide-2 (NS2) polypeptide; c) an HCVnon-structural polypeptide-4A (NS4A) polypeptide; d) an HCVnon-structural polypeptide-4B (NS4B) polypeptide; e) an HCVnon-structural polypeptide-5A (NS5A) polypeptide; f) an HCVnon-structural polypeptide-5B (NS5B) polypeptide; g) an HCV corepolypeptide; and h) an HCV p7 polypeptide.

Aspect 3. The immunogenic composition of aspect 1 or aspect 2, wherein:a) the HCV E2 polypeptide is derived from an HCV of genotype 1, 2, 3, 4,5, 6, or 7; and b) the HCV E1 polypeptide is derived from an HCV ofgenotype 1, 2, 3, 4, 5, 6, or 7.

Aspect 4. The immunogenic composition of aspect 1, wherein the HCV E2polypeptide comprises an amino acid sequence having at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to an E2 polypeptide depicted in one of FIG. 1A-1C, FIG. 2A-2C,FIG. 3A-3C, and FIG. 4A-4B.

Aspect 5. The immunogenic composition of any one of aspects 1-4, whereinthe T-cell epitope polypeptide has a length of from about 10 amino acidsto about 3000 amino acids.

Aspect 6. The immunogenic composition of aspects 1-4, wherein the T-cellepitope polypeptide has a length of from about 10 amino acids to about50 amino acids, from about 100 amino acids to about 230 amino acids,from about 230 amino acids to about 550 amino acids, from about 29 aminoacids to about 780 amino acids, from about 100 amino acids to about 780amino acids, from about 550 amino acids to about 780 amino acids, fromabout 780 amino acids to 1985 amino acids, or from about 780 amino acidsto about 2000 amino acids.

Aspect 7. The immunogenic composition of any one of aspects 1-6, whereinthe T-cell epitope polypeptide comprises one or more T cell epitopespresent in an HCV NS3 polypeptide.

Aspect 8. The immunogenic composition of aspect 7, wherein the T-cellepitope polypeptide comprises an amino acid sequence having at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence of one of TP29, TP50, TP52,TP70, TP100, TP171, TP228, TP553, TP778, and TP1985.

Aspect 9. The immunogenic composition of any one of aspects 1-8, whereinthe T-cell epitope polypeptide comprises one or more T cell epitopespresent in: a) cholera toxin or toxoid; and/or b) tetanus toxin ortoxoid; and/or c) diphtheria toxin or toxoid; and/or d) CRM197.

Aspect 10. The immunogenic composition of any one of aspects 1-8,wherein the composition comprises a polypeptide comprising one or more Tcell epitopes present in: a) cholera toxin or toxoid; and/or b) tetanustoxin or toxoid; and/or c) diphtheria toxin or toxoid; and/or d) CRM197.

Aspect 11. The immunogenic composition of any one of aspects 1-10,wherein the E2 polypeptide and/or the E1 polypeptide lacks a C-terminaltransmembrane domain.

Aspect 12. The immunogenic composition of any one of aspects 1-11,wherein the HCV E2 polypeptide and the HCV E1 polypeptide are derivedfrom an HCV of the same genotype.

Aspect 13. The immunogenic composition of any one of aspects 1-11,wherein the HCV E2 polypeptide and the HCV E1 polypeptide are derivedfrom an HCV of different genotypes.

Aspect 14. The immunogenic composition of any one of aspects 1-13,wherein the HCV E1 polypeptide comprises an amino acid sequence havingat least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to an E1 polypeptide depicted in FIG. 1A-1C, FIG.2A-2C, FIG. 3A-3C, and FIG. 4A-4B.

Aspect 15. The immunogenic composition of aspect 1, wherein the HCVE1/E2 heterodimeric polypeptide comprises:

a) an HCV E1 polypeptide; and b) a modified E2 polypeptide comprising,in order from N-terminus to C-terminus: i) from 1 to 6 heterologousamino acids, wherein the from 1 to 6 heterologous amino acids areC-terminal to a site of proteolytic cleavage in a proteolyticallycleavable linker; and ii) an HCV E2 polypeptide; or

a) an HCV E2 polypeptide; and b) a modified E1 polypeptide comprising,in order from N-terminus to C-terminus: i) from 1 to 6 heterologousamino acids, wherein the from 1 to 6 heterologous amino acids areC-terminal to a site of proteolytic cleavage in a proteolyticallycleavable linker; and ii) an HCV E1 polypeptide; or

a) an HCV E1 polypeptide; and b) a modified E2 polypeptide comprising,in order from N-terminus to C-terminus: i) an HCV E2 polypeptide; andii) from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are N-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; or

a) an HCV E2 polypeptide; and b) a modified E1 polypeptide comprising,in order from N-terminus to C-terminus: i) an HCV E1 polypeptide; andii) from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are N-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker.

Aspect 16. The immunogenic composition of aspect 15, wherein: a) thefrom 1 to 6 heterologous amino acids at the N-terminus of the modifiedE2 polypeptide or the modified E1 polypeptide are Gly-Pro, Ser, Gly, orGly-Ser; or b) the from 1 to 6 heterologous amino acids at theC-terminus of the modified E2 polypeptide or the modified E1 polypeptideare LEVLFQ (SEQ ID NO:76), ENLYYFQ (SEQ ID NO:83), LVPR (SEQ ID NO:78),I(E/D)GR (SEQ ID NO:90), or DDDDK (SEQ ID NO:77).

Aspect 17. The immunogenic composition of any one of aspects 1-16,comprising an adjuvant.

Aspect 18. The immunogenic composition of aspect 17, wherein theadjuvant comprises MF59; alum; poly(DL-lactide co-glycolide); a CpGoligonucleotide; keyhole limpet hemocyanin; a suspension of liposomescomprising 3′-O-desacyl-4′-monophosphoryl lipid A (MPL) and Quillajasaponaria 21 (QS21); AS01; or alum+MPL.

Aspect 19. The immunogenic composition of any one of aspects 1-18,wherein the T-cell epitope polypeptide comprises an amino acid sequencehaving at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%,amino acid sequence identity to a polypeptide depicted in any one ofFIGS. 9A, 9B, 10A-10D, and 11A-11N.

Aspect 20. A method of inducing an immune response in an individual to ahepatitis C virus (HCV) polypeptide, the method comprising administeringto the individual an effective amount of the immunogenic composition ofany one of aspects 1-19.

Aspect 21. The method of aspect 20, wherein said administering is byintramuscular administration.

Aspect 22. The method of aspect 20, wherein said administering is bysubcutaneous administration.

Aspect 23. An immunogenic composition comprising: a) a hepatitis C virus(HCV) T-cell epitope polypeptide comprising a T-cell epitope present inan HCV protein other than E1 and E2; and b) a pharmaceuticallyacceptable excipient.

Aspect 24. The immunogenic composition of aspect 23, wherein the HCVT-cell epitope polypeptide comprises one or more T cell epitopes presentin one or more of:

a) an HCV non-structural polypeptide-3 (NS3) polypeptide;

b) an HCV non-structural polypeptide-2 (NS2) polypeptide;

c) an HCV non-structural polypeptide-4A (NS4A) polypeptide;

d) an HCV non-structural polypeptide-4B (NS4B) polypeptide;

e) an HCV non-structural polypeptide-5A (NS5A) polypeptide;

an HCV non-structural polypeptide-5B (NS5B) polypeptide;

g) an HCV core polypeptide; and

h) an HCV p7 polypeptide.

Aspect 25. The immunogenic composition of aspect 23, wherein the HCVT-cell epitope polypeptide has a length of from about 10 amino acids toabout 50 amino acids, from about 100 amino acids to about 230 aminoacids, from about 230 amino acids to about 550 amino acids, from about29 amino acids to about 780 amino acids, from about 100 amino acids toabout 780 amino acids, from about 550 amino acids to about 780 aminoacids, from about 780 amino acids to 1985 amino acids, or from about 780amino acids to about 2000 amino acids.

Aspect 26. The immunogenic composition of any one of aspects 23-25,wherein the HCV T-cell epitope polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide.

Aspect 27. The immunogenic composition any one of aspects 23-26, whereinthe HCV T-cell epitope polypeptide comprises an amino acid sequencehaving at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%,amino acid sequence identity to the amino acid sequence of one of TP29,TP50, TP52, TP70, TP100, TP171, TP228, TP553, TP778, and TP1985.

Aspect 28. The immunogenic composition of any one of aspects 23-27,wherein the HCV T-cell epitope polypeptide comprises one or more T cellepitopes present in:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 29. The immunogenic composition of any one of aspects 23-27,wherein the composition comprises a polypeptide comprising one or more Tcell epitopes present in: a) cholera toxin or toxoid; and/or b) tetanustoxin or toxoid; and/or c) diphtheria toxin or toxoid; and/or d) CRM197.

Aspect 30. The immunogenic composition of any one of aspects 23-29,comprising an adjuvant.

Aspect 31. The immunogenic composition of aspect 30, wherein theadjuvant comprises MF59; alum; poly(DL-lactide co-glycolide); a CpGoligonucleotide; keyhole limpet hemocyanin; a suspension of liposomescomprising 3′-O-desacyl-4′-monophosphoryl lipid A (MPL) and Quillajasaponaria 21 (QS21); AS01; or alum+MPL.

Aspect 32. The immunogenic composition of any one of aspects 23-31,wherein the HCV T-cell polypeptide comprises an amino acid sequencehaving at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%,amino acid sequence identity to a polypeptide depicted in any one ofFIGS. 9A, 9B, 10A-10D, and 11A-11N.

Aspect 33. The immunogenic composition of any one of aspects 23-32,comprising two or more different HCV T-cell epitope polypeptides.

Aspect 34. A method of inducing an immune response in an individual, themethod comprising administering to the individual an effective amount ofthe immunogenic composition of any one of aspects 23-33.

Aspect 35. The method of aspect 34, wherein said administering comprisessubcutaneous administration or intramuscular administration.

Aspect 36. An immunogenic composition comprising: a) a hepatitis C virus(HCV) E2 polypeptide; b) a T-cell epitope polypeptide comprising aT-cell epitope present in an HCV protein other than E1 and E2; and c) apharmaceutically acceptable carrier.

Aspect 37. The immunogenic composition of aspect 36, wherein the T-cellepitope polypeptide comprises one or more T cell epitopes present in oneor more of:

a) an HCV non-structural polypeptide-3 (NS3) polypeptide;

b) an HCV non-structural polypeptide-2 (NS2) polypeptide;

c) an HCV non-structural polypeptide-4A (NS4A) polypeptide;

d) an HCV non-structural polypeptide-4B (NS4B) polypeptide;

e) an HCV non-structural polypeptide-5A (NS5A) polypeptide;

f) an HCV non-structural polypeptide-5B (NS5B) polypeptide;

g) an HCV core polypeptide; and

h) an HCV p7 polypeptide.

Aspect 38. The immunogenic composition of aspect 36 or aspect 37,wherein the HCV E2 polypeptide is derived from an HCV of genotype 1, 2,3, 4, 5, 6, or 7.

Aspect 39. The immunogenic composition of aspect 36, wherein the HCV E2polypeptide comprises an amino acid sequence having at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to an E2 polypeptide depicted in one of FIG. 1A-1C, FIG. 2A-2C,FIG. 3A-3C, and FIG. 4A-4B.

Aspect 40. The immunogenic composition of any one of aspects 1-4,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 3000 amino acids.

Aspect 41. The immunogenic composition of aspects 36-40, wherein theT-cell epitope polypeptide has a length of from about 10 amino acids toabout 50 amino acids, from about 100 amino acids to about 230 aminoacids, from about 230 amino acids to about 550 amino acids, from about29 amino acids to about 780 amino acids, from about 100 amino acids toabout 780 amino acids, from about 550 amino acids to about 780 aminoacids, from about 780 amino acids to 1985 amino acids, or from about 780amino acids to about 2000 amino acids.

Aspect 42. The immunogenic composition of any one of aspects 36-41,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide.

Aspect 43. The immunogenic composition of aspect 42, wherein the T-cellepitope polypeptide comprises an amino acid sequence having at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, at least 99%, or 100%, amino acidsequence identity to the amino acid sequence of one of TP29, TP50, TP52,TP70, TP100, TP171, TP228, TP553, TP778, and TP1985.

Aspect 44. The immunogenic composition of any one of aspects 36-43,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in:

a) cholera toxin or toxoid; and/or

b) tetanus toxin or toxoid; and/or

c) diphtheria toxin or toxoid; and/or

d) CRM197.

Aspect 45. The immunogenic composition of any one of aspects 36-43,wherein the composition comprises a polypeptide comprising one or more Tcell epitopes present in: a) cholera toxin or toxoid; and/or b) tetanustoxin or toxoid; and/or c) diphtheria toxin or toxoid; and/or d) CRM197.

Aspect 46. The immunogenic composition of any one of aspects 36-45,wherein the E2 polypeptide lacks a C-terminal transmembrane domain.

Aspect 47. The immunogenic composition of any one of aspects 36-46,wherein the HCV E2 polypeptide is:

a) a modified E2 polypeptide comprising, in order from N-terminus toC-terminus: i) from 1 to 6 heterologous amino acids, wherein the from 1to 6 heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and ii) an HCV E2polypeptide; or

b) a modified E2 polypeptide comprising, in order from N-terminus toC-terminus: i) an HCV E2 polypeptide; and ii) from 1 to 6 heterologousamino acids, wherein the from 1 to 6 heterologous amino acids areN-terminal to a site of proteolytic cleavage in a proteolyticallycleavable linker.

Aspect 48. The immunogenic composition of aspect 47, wherein: a) thefrom 1 to 6 heterologous amino acids at the N-terminus of the modifiedE1 polypeptide are Gly-Pro, Ser, Gly, or Gly-Ser; or b) the from 1 to 6heterologous amino acids at the C-terminus of the modified E1polypeptide are LEVLFQ (SEQ ID NO:76), ENLYYFQ (SEQ ID NO:83), LVPR (SEQID NO:78), I(E/D)GR (SEQ ID NO:90), or DDDDK (SEQ ID NO:77).

Aspect 49. The immunogenic composition of any one of aspects 36-48,comprising an adjuvant.

Aspect 50. The immunogenic composition of aspect 49, wherein theadjuvant comprises MF59; alum; poly(DL-lactide co-glycolide); a CpGoligonucleotide; keyhole limpet hemocyanin; a suspension of liposomescomprising 3′-O-desacyl-4′-monophosphoryl lipid A (MPL) and Quillajasaponaria 21 (QS21); AS01; or a combination of alum and MPL.

Aspect 51. The immunogenic composition of any one of aspects 36-50,wherein the T-cell epitope polypeptide comprises an amino acid sequencehaving at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%,amino acid sequence identity to a polypeptide depicted in any one ofFIGS. 9A, 9B, 10A-10D, and 11A-11N.

Aspect 52. A method of inducing an immune response in an individual to ahepatitis C virus (HCV) polypeptide, the method comprising administeringto the individual an effective amount of the immunogenic composition ofany one of aspects 36-51.

Aspect 53. The method of aspect 52, wherein said administering is byintramuscular administration.

Aspect 54. The method of aspect 52, wherein said administering is bysubcutaneous administration.

Aspect 55. An immunogenic composition comprising: a) a hepatitis C virus(HCV) E1 polypeptide; b) a T-cell epitope polypeptide comprising aT-cell epitope present in an HCV protein other than E1 and E2; and c) apharmaceutically acceptable carrier.

Aspect 56. The immunogenic composition of aspect 55, wherein the T-cellepitope polypeptide comprises one or more T cell epitopes present in oneor more of:

a) an HCV non-structural polypeptide-3 (NS3) polypeptide;

b) an HCV non-structural polypeptide-2 (NS2) polypeptide;

c) an HCV non-structural polypeptide-4A (NS4A) polypeptide;

d) an HCV non-structural polypeptide-4B (NS4B) polypeptide;

e) an HCV non-structural polypeptide-5A (NS5A) polypeptide;

an HCV non-structural polypeptide-5B (NS5B) polypeptide;

g) an HCV core polypeptide; and

h) an HCV p7 polypeptide.

Aspect 57. The immunogenic composition of aspect 55 or aspect 56,wherein the HCV E1 polypeptide is derived from an HCV of genotype 1, 2,3, 4, 5, 6, or 7.

Aspect 58. The immunogenic composition of aspect 55, wherein the HCV E1polypeptide comprises an amino acid sequence having at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, at least 99%, or 100%, amino acid sequenceidentity to an E1 polypeptide depicted in one of FIG. 1A-1C, FIG. 2A-2C,FIG. 3A-3C, and FIG. 4A-4B.

Aspect 59. The immunogenic composition of any one of aspects 55-58,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 3000 amino acids.

Aspect 60. The immunogenic composition of any one of aspects 55-58,wherein the T-cell epitope polypeptide has a length of from about 10amino acids to about 50 amino acids, from about 100 amino acids to about230 amino acids, from about 230 amino acids to about 550 amino acids,from about 29 amino acids to about 780 amino acids, from about 100 aminoacids to about 780 amino acids, from about 550 amino acids to about 780amino acids, from about 780 amino acids to 1985 amino acids, or fromabout 780 amino acids to about 2000 amino acids.

Aspect 61. The immunogenic composition of any one of aspects 55-60,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in an HCV NS3 polypeptide.

Aspect 62. The immunogenic composition of any one of aspects 55-60,wherein the T-cell epitope polypeptide comprises an amino acid sequencehaving at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%,amino acid sequence identity to the amino acid sequence of one of TP29,TP50, TP52, TP70, TP100, TP171, TP228, TP553, TP778, and TP1985.

Aspect 63. The immunogenic composition of any one of aspects 55-62,wherein the T-cell epitope polypeptide comprises one or more T cellepitopes present in: a) cholera toxin or toxoid; and/or b) tetanus toxinor toxoid; and/or c) diphtheria toxin or toxoid; and/or d) CRM197.

Aspect 64. The immunogenic composition of any one of aspects 55-62,wherein the composition comprises a polypeptide comprising one or more Tcell epitopes present in: a) cholera toxin or toxoid; and/or b) tetanustoxin or toxoid; and/or c) diphtheria toxin or toxoid; and/or d) CRM197.

Aspect 65. The immunogenic composition of any one of aspects 55-64,wherein the E1 polypeptide lacks a C-terminal transmembrane domain.

Aspect 66. The immunogenic composition of any one of aspects 55-64,wherein the HCV E1 polypeptide is:

a) a modified E1 polypeptide comprising, in order from N-terminus toC-terminus: i) from 1 to 6 heterologous amino acids, wherein the from 1to 6 heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and ii) an HCV E1polypeptide; or

b) a modified E1 polypeptide comprising, in order from N-terminus toC-terminus: i) an HCV E1 polypeptide; and ii) from 1 to 6 heterologousamino acids, wherein the from 1 to 6 heterologous amino acids areN-terminal to a site of proteolytic cleavage in a proteolyticallycleavable linker.

Aspect 67. The immunogenic composition of aspect 66, wherein: a) thefrom 1 to 6 heterologous amino acids at the N-terminus of the modifiedE1 polypeptide are Gly-Pro, Ser, Gly, or Gly-Ser; or b) the from 1 to 6heterologous amino acids at the C-terminus of the modified E1polypeptide are LEVLFQ (SEQ ID NO:76), ENLYYFQ (SEQ ID NO:83), LVPR (SEQID NO:78), I(E/D)GR (SEQ ID NO:90), or DDDDK (SEQ ID NO:77).

Aspect 68. The immunogenic composition of any one of aspects 65-67,comprising an adjuvant.

Aspect 69. The immunogenic composition of aspect 68, wherein theadjuvant comprises MF59; alum; poly(DL-lactide co-glycolide); a CpGoligonucleotide; keyhole limpet hemocyanin; a suspension of liposomescomprising 3′-O-desacyl-4′-monophosphoryl lipid A (MPL) and Quillajasaponaria 21 (QS21); AS01; or a combination of alum and MPL.

Aspect 70. The immunogenic composition of any one of aspects 55-69,wherein the T-cell epitope polypeptide comprises an amino acid sequencehaving at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%,amino acid sequence identity to a polypeptide depicted in any one ofFIGS. 9A, 9B, 10A-10D, and 11A-11N.

Aspect 71. A method of inducing an immune response in an individual to ahepatitis C virus (HCV) polypeptide, the method comprising administeringto the individual an effective amount of the immunogenic composition ofany one of aspects 55-70.

Aspect 72. The method of aspect 71, wherein said administering is byintramuscular administration.

Aspect 73. The method of aspect 71, wherein said administering is bysubcutaneous administration.

Aspect 74. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP29 and having a length of29 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 75. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP50 and having a length of50 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 76. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP52 and having a length of52 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 77. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP70 and having a length of70 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 78. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP100 and having a lengthof 100 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 79. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP171 and having a lengthof 171 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 80. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP228 and having a lengthof 228 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 81. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP553 and having a lengthof 553 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 82. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP778 and having a lengthof 778 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 83. An immunogenic composition comprising:

a) a hepatitis C virus (HCV) heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) a modified HCV E2 polypeptide comprising a heterologous        Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus of an        HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising an amino acid sequence havingat least 95% amino acid sequence identity to TP1985 and having a lengthof 1985 amino acids; and

c) a pharmaceutically acceptable carrier.

Aspect 84. The immunogenic composition of any one of aspects 74-83,comprising an adjuvant.

Aspect 85. The immunogenic composition of aspect 83, wherein theadjuvant comprises MF59; alum; poly(DL-lactide co-glycolide); a CpGoligonucleotide; keyhole limpet hemocyanin; a suspension of liposomescomprising 3′-O-desacyl-4′-monophosphoryl lipid A (MPL) and Quillajasaponaria 21 (QS21); AS01; or a combination of alum and MPL.

Aspect 86. The immunogenic composition of any one of aspects 74-83,wherein the HCV E1 and E2 polypeptides are a mixture of HCV genotype 1and HCV genotype 3.

Aspect 87. The immunogenic composition of any one of aspects 74-83,wherein the HCV E1 and E2 polypeptides are a mixture of HCV genotype 1,HCV genotype 2, and HCV genotype 3.

Aspect 88. A method of inducing an immune response to HCV in anindividual, the method comprising administering to the individual aneffective amount of a nucleic acid immunogenic composition comprising:a) one or more nucleic acids comprising nucleotide sequences encodingone or more of: i) an HCV E1/E2 heterodimer; ii) an HCV E1 polypeptide;iii) an HCV E2 polypeptide; and iv) a T-cell epitope polypeptidecomprising a T-cell epitope present in an HCV protein other than E1 andE2; and b) a pharmaceutically acceptable carrier.

Aspect 89. The method of aspect 88, wherein the one or more nucleicacids are recombinant expression vectors.

Aspect 90. The method of aspect 89, wherein the one or more recombinantexpression vectors are recombinant viral vectors.

Aspect 91. The method of aspect 90, wherein the one or more recombinantviral vectors are packaged into viral particles.

Aspect 92. The method of aspect 88, wherein the one or more nucleicacids are present within non-pathogenic bacteria.

Aspect 93. The method of aspect 89, comprising administering: a) a firstrecombinant expression vector comprising nucleotide sequences encodingthe HCV E1/E2 heterodimer, and HCV E1 polypeptide, or an HCV E2polypeptide; and b) a second recombinant expression vector comprisingnucleotide sequences encoding the T-cell epitope polypeptide.

Aspect 94. The method of aspect 89, comprising administering: a) a firstrecombinant expression vector comprising nucleotide sequences encodingthe HCV E1/E2 heterodimer; and b) a second recombinant expression vectorcomprising nucleotide sequences encoding the T-cell epitope polypeptide.

Aspect 95. The method of aspect 94, wherein the first recombinantexpression vector and the second recombinant expression vector are twodifferent recombinant virus-based vectors.

Aspect 96. The method of aspect 89, wherein said recombinant expressionvector is a recombinant modified vaccinia Ankara vector.

Aspect 97. The method of aspect 89, wherein said recombinant expressionvector is a recombinant replication-defective adenovirus.

Aspect 98. The method of aspect 88, comprising administering apolypeptide immunogenic composition, wherein the second immunogeniccomposition comprises:

a) a hepatitis C virus (HCV) E1/E2 heterodimeric polypeptide comprising:

-   -   i) an HCV E1 polypeptide; and    -   ii) an HCV E2 polypeptide;

b) a T-cell epitope polypeptide comprising a T-cell epitope present inan HCV protein other than E1 and E2; and

c) a pharmaceutically acceptable carrier.

Aspect 99. The method of aspect 98, wherein the nucleic acid immunogeniccomposition and the polypeptide immunogenic composition are administeredsubstantially simultaneously.

Aspect 100. The method of aspect 98, wherein the nucleic acidimmunogenic composition is administered as a prime administration, andthe polypeptide immunogenic composition is administered as a boostadministration.

Aspect 101. The method of aspect 98, wherein the polypeptide immunogeniccomposition is administered as a prime administration, and the nucleicacid immunogenic composition is administered as a boost administration.

Aspect 102. The method of any one of aspects 88-101, wherein saidadministering is by intramuscular administration.

Aspect 103. The method of any one of aspects 88-101, wherein saidadministering is by subcutaneous administration.

Aspect 104. An immunogenic composition comprising:

a) one or more nucleic acids comprising nucleotide sequences encodingone or more of: i) an HCV E1/E2 heterodimer; ii) an HCV E1 polypeptide;iii) an HCV E2 polypeptide; and iv) a T-cell epitope polypeptidecomprising a T-cell epitope present in an HCV protein other than E1 andE2; and

b) a pharmaceutically acceptable carrier.

Aspect 105. The immunogenic composition of aspect 104, wherein the oneor more nucleic acids are recombinant expression vectors.

Aspect 106. The immunogenic composition of aspect 105, wherein the oneor more recombinant expression vectors are recombinant viral vectors.

Aspect 107. The immunogenic composition of aspect 106, wherein the oneor more recombinant viral vectors are packaged into viral particles.

Aspect 108. The immunogenic composition of aspect 104, wherein the oneor more nucleic acids are present within non-pathogenic bacteria.

Aspect 109. The immunogenic composition of aspect 104, wherein the oneor more nucleic acids are DNA.

Aspect 110. The immunogenic composition of aspect 104, wherein the oneor more nucleic acids are RNA.

Aspect 111. An immunogenic composition comprising:

a) one or more nucleic acids comprising nucleotide sequences encodingone or more the polypeptides present in an immunogenic composition ofany one of aspects 1-19, aspects 23-33, aspects 36-51, aspects 55-70, oraspects 74-83; and

b) a pharmaceutically acceptable carrier.

Aspect 112. The immunogenic composition of aspect 111, wherein the oneor more nucleic acids are recombinant expression vectors.

Aspect 113. The immunogenic composition of aspect 112, wherein the oneor more recombinant expression vectors are recombinant viral vectors.

Aspect 114. The immunogenic composition of aspect 113, wherein the oneor more recombinant viral vectors are packaged into viral particles.

Aspect 115. The immunogenic composition of aspect 111, wherein the oneor more nucleic acids are present within non-pathogenic bacteria.

Aspect 116. The immunogenic composition of aspect 111, wherein the oneor more nucleic acids are DNA.

Aspect 117. The immunogenic composition of aspect 111, wherein the oneor more nucleic acids are RNA.

Aspect 118. A method of inducing an immune response to HCV in anindividual, the method comprising administering to the individual aneffective amount of an immunogenic composition of any one of aspects111-117.

Aspect 119. The method of aspect 118, wherein the one or more nucleicacids are present in one or more recombinant viral expression vectors.

Aspect 120. The method of aspect 119, wherein the one or morerecombinant expression vectors comprise a recombinant modified vacciniaAnkara vector.

Aspect 121. The method of aspect 119, wherein the one or morerecombinant expression vectors comprise a recombinantreplication-defective adenovirus.

Aspect 122. The method of aspect 118, wherein the one or more nucleicacids comprise a first recombinant expression vector and a secondrecombinant expression vector, wherein first recombinant expressionvector and the second recombinant expression vector are two differentrecombinant virus-based vectors.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An immunogenic composition comprising as separateentities: a) a hepatitis C virus (HCV) E1/E2 heterodimeric polypeptidecomprising: i) an HCV E1 polypeptide; and ii) an HCV E2 polypeptide; b)a T-cell epitope polypeptide comprising a T-cell epitope present in anHCV protein other than E1 and E2; and c) a pharmaceutically acceptablecarrier.
 2. The immunogenic composition of claim 1, wherein the T-cellepitope polypeptide comprises one or more T cell epitopes present in oneor more of: a) an HCV non-structural polypeptide-3 (NS3) polypeptide; b)an HCV non-structural polypeptide-2 (NS2) polypeptide; c) an HCVnon-structural polypeptide-4A (NS4A) polypeptide; d) an HCVnon-structural polypeptide-4B (NS4B) polypeptide; e) an HCVnon-structural polypeptide-5A (NS5A) polypeptide; f) an HCVnon-structural polypeptide-5B (NS5B) polypeptide; g) an HCV corepolypeptide; and h) an HCV p7 polypeptide.
 3. The immunogeniccomposition of claim 1, wherein: a) the HCV E2 polypeptide is derivedfrom an HCV of major genotype 1, 2, 3, 4, 5, 6, or 7; and b) the HCV E1polypeptide is derived from an HCV of major genotype 1, 2, 3, 4, 5, 6,or
 7. 4. The immunogenic composition of claim 1, wherein the T-cellepitope polypeptide has a length of from about 10 amino acids to about3000 amino acids.
 5. The immunogenic composition of claim 1, wherein theT-cell epitope polypeptide comprises one or more T cell epitopes presentin an HCV NS3 polypeptide.
 6. The immunogenic composition of claim 5,wherein the T-cell epitope polypeptide comprises an amino acid sequencehaving at least 20% amino acid sequence identity to the amino acidsequence of one of a TP29 polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:94, a TP50 polypeptide comprising the amino acidsequence set forth in SEQ ID NO:99, a TP52 polypeptide comprising theamino acid sequence set forth in SEQ ID NO:95, a TP70 polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:96, a TP100polypeptide comprising the amino acid sequence set forth in SEQ IDNO:97, a TP171 polypeptide comprising the amino acid sequence set forthin SEQ ID NO:89, a TP228 polypeptide comprising the amino acid sequenceset forth in SEQ ID NO:81, a TP553 polypeptide comprising the amino acidsequence set forth in SEQ ID NO:100, a TP778 polypeptide comprising theamino acid sequence set forth in SEQ ID NO:101, and a TP1985 polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:102.
 7. Theimmunogenic composition of claim 1, wherein the T-cell epitopepolypeptide comprises one or more T cell epitopes present in: a) choleratoxin or toxoid; and/or b) tetanus toxin or toxoid; and/or c) diphtheriatoxin or toxoid; and/or d) CRM197.
 8. The immunogenic composition ofclaim 1, wherein the composition comprises a polypeptide comprising oneor more T cell epitopes present in: a) cholera toxin or toxoid; and/orb) tetanus toxin or toxoid; and/or c) diphtheria toxin or toxoid; and/ord) CRM197.
 9. The immunogenic composition of claim 1, wherein the E2polypeptide and/or the E1 polypeptide lacks a C-terminal transmembranedomain.
 10. The immunogenic composition of claim 1, wherein the HCVE1/E2 heterodimeric polypeptide comprises: a) an HCV E1 polypeptide; andb) a modified E2 polypeptide comprising, in order from N-terminus toC-terminus: i) from 1 to 6 heterologous amino acids, wherein the from 1to 6 heterologous amino acids are C-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; and ii) an HCV E2polypeptide; or a) an HCV E2 polypeptide; and b) a modified E1polypeptide comprising, in order from N-terminus to C-terminus: i) from1 to 6 heterologous amino acids, wherein the from 1 to 6 heterologousamino acids are C-terminal to a site of proteolytic cleavage in aproteolytically cleavable linker; and ii) an HCV E1 polypeptide; or a)an HCV E1 polypeptide; and b) a modified E2 polypeptide comprising, inorder from N-terminus to C-terminus: i) an HCV E2 polypeptide; and ii)from 1 to 6 heterologous amino acids, wherein the from 1 to 6heterologous amino acids are N-terminal to a site of proteolyticcleavage in a proteolytically cleavable linker; or a) an HCV E2polypeptide; and b) a modified E1 polypeptide comprising, in order fromN-terminus to C-terminus: i) an HCV E1 polypeptide; and ii) from 1 to 6heterologous amino acids, wherein the from 1 to 6 heterologous aminoacids are N-terminal to a site of proteolytic cleavage in aproteolytically cleavable linker.
 11. The immunogenic composition ofclaim 10, wherein: a) the from 1 to 6 heterologous amino acids at theN-terminus of the modified E2 polypeptide or the modified E1 polypeptideare Gly-Pro, Ser, Gly, or Gly-Ser; or b) the from 1 to 6 heterologousamino acids at the C-terminus of the modified E2 polypeptide or themodified E1 polypeptide are LEVLFQ as set forth in SEQ ID NO:76, ENLYYFQas set forth in SEQ ID NO:83, LVPR as set forth in SEQ ID NO:78,I(E/D)GR as set forth in SEQ ID NO:90, or DDDDK as set forth in SEQ IDNO:77.
 12. The immunogenic composition of claim 1, comprising anadjuvant.
 13. The immunogenic composition of claim 12, wherein theadjuvant comprises MF59; alum; poly(DL-lactide co-glycolide); a CpGoligonucleotide; keyhole limpet hemocyanin; or a suspension of liposomescomprising 3′-O-desacyl-4′-monophosphoryl lipid A (MPL) and Quillajasaponaria 21 (QS21); AS01; or a mixture of alum and MPL.
 14. A method ofinducing an immune response in an individual to a hepatitis C virus(HCV) polypeptide, the method comprising administering to theindividual: a) an effective amount of the immunogenic composition ofclaim 1; or b) one or more nucleic acids comprising nucleotide sequencesencoding the HCV E1/E2 heterodimeric polypeptide and the T-cell epitopepolypeptide of claim
 1. 15. An immunogenic composition comprising: a1) ahepatitis C virus (HCV) heterodimeric polypeptide comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aheterologous Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus ofan HCV E2 polypeptide; b1) a T-cell epitope polypeptide comprising anamino acid sequence having at least 95% amino acid sequence identity toa TP29 polypeptide comprising the amino acid sequence set forth in SEQID NO:94 and having a length of 29 amino acids; and c1) apharmaceutically acceptable carrier; or a2) a hepatitis C virus (HCV)heterodimeric polypeptide comprising: i) an HCV E1 polypeptide; and ii)a modified HCV E2 polypeptide comprising a heterologous Gly-Pro,Gly-Ser, Gly, or Ser appended to the N-terminus of an HCV E2polypeptide; b2) a T-cell epitope polypeptide comprising an amino acidsequence having at least 95% amino acid sequence identity to a TP50polypeptide comprising the amino acid sequence set forth in SEQ ID NO:99and having a length of 50 amino acids; and c2) a pharmaceuticallyacceptable carrier; or a3) a hepatitis C virus (HCV) heterodimericpolypeptide comprising: i) an HCV E1 polypeptide; and ii) a modified HCVE2 polypeptide comprising a heterologous Gly-Pro, Gly-Ser, Gly, or Serappended to the N-terminus of an HCV E2 polypeptide; b3) a T-cellepitope polypeptide comprising an amino acid sequence having at least95% amino acid sequence identity to a TP52 polypeptide comprising theamino acid sequence set forth in SEQ ID NO:95 and having a length of 52amino acids; and c3) a pharmaceutically acceptable carrier; or a4) ahepatitis C virus (HCV) heterodimeric polypeptide comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aheterologous Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus ofan HCV E2 polypeptide; b4) a T-cell epitope polypeptide comprising anamino acid sequence having at least 95% amino acid sequence identity toa TP70 polypeptide comprising the amino acid sequence set forth in SEQID NO:96 and having a length of 70 amino acids; and c4) apharmaceutically acceptable carrier; or a5) a hepatitis C virus (HCV)heterodimeric polypeptide comprising: i) an HCV E1 polypeptide; and ii)a modified HCV E2 polypeptide comprising a heterologous Gly-Pro,Gly-Ser, Gly, or Ser appended to the N-terminus of an HCV E2polypeptide; b5) a T-cell epitope polypeptide comprising an amino acidsequence having at least 95% amino acid sequence identity to a TP100polypeptide comprising the amino acid sequence set forth in SEQ ID NO:97and having a length of 100 amino acids; and c5) a pharmaceuticallyacceptable carrier; or a6) a hepatitis C virus (HCV) heterodimericpolypeptide comprising: i) an HCV E1 polypeptide; and ii) a modified HCVE2 polypeptide comprising a heterologous Gly-Pro, Gly-Ser, Gly, or Serappended to the N-terminus of an HCV E2 polypeptide; b6) a T-cellepitope polypeptide comprising an amino acid sequence having at least95% amino acid sequence identity to a TP171 polypeptide comprising theamino acid sequence set forth in SEQ ID NO:89 and having a length of 171amino acids; and c6) a pharmaceutically acceptable carrier; or a7) ahepatitis C virus (HCV) heterodimeric polypeptide comprising: i) an HCVE1 polypeptide; and ii) a modified HCV E2 polypeptide comprising aheterologous Gly-Pro, Gly-Ser, Gly, or Ser appended to the N-terminus ofan HCV E2 polypeptide; b7) a T-cell epitope polypeptide comprising anamino acid sequence having at least 95% amino acid sequence identity toa TP228 polypeptide comprising the amino acid sequence set forth in SEQID NO:81 and having a length of 228 amino acids; and c7) apharmaceutically acceptable carrier; or a8) a hepatitis C virus (HCV)heterodimeric polypeptide comprising: i) an HCV E1 polypeptide; and ii)a modified HCV E2 polypeptide comprising a heterologous Gly-Pro,Gly-Ser, Gly, or Ser appended to the N-terminus of an HCV E2polypeptide; b8) a T-cell epitope polypeptide comprising an amino acidsequence having at least 95% amino acid sequence identity to a TP553polypeptide comprising the amino acid sequence set forth in SEQ IDNO:100 and having a length of 553 amino acids; and c8) apharmaceutically acceptable carrier; or a9) a hepatitis C virus (HCV)heterodimeric polypeptide comprising: i) an HCV E1 polypeptide; and ii)a modified HCV E2 polypeptide comprising a heterologous Gly-Pro,Gly-Ser, Gly, or Ser appended to the N-terminus of an HCV E2polypeptide; b99) a T-cell epitope polypeptide comprising an amino acidsequence having at least 95% amino acid sequence identity to a TP778polypeptide comprising the amino acid sequence set forth in SEQ IDNO:101 and having a length of 778 amino acids; and c) a pharmaceuticallyacceptable carrier; or a10) a hepatitis C virus (HCV) heterodimericpolypeptide comprising: i) an HCV E1 polypeptide; and ii) a modified HCVE2 polypeptide comprising a heterologous Gly-Pro, Gly-Ser, Gly, or Serappended to the N-terminus of an HCV E2 polypeptide; b10) a T-cellepitope polypeptide comprising an amino acid sequence having at least95% amino acid sequence identity to a TP1985 polypeptide comprising theamino acid sequence set forth in SEQ ID NO:102 and having a length of1985 amino acids; and c10) a pharmaceutically acceptable carrier. 16.The immunogenic composition of claim 15, comprising an adjuvant.